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Recent studies have demonstrated that visfatin participates in the regulation of female reproduction. Due to the lack of data concerning the level of visfatin in the ovarian follicles of pigs, one of the most economically important livestock species, the aim of this study was to investigate the expression and localisation of visfatin and the follicular fluid concentration in the ovarian follicles of prepubertal and mature gilts. We also aimed to examine the in vitro effects of gonadotropins (LH, FSH), insulin, progesterone (P 4 ), oestradiol (E 2 ), prostaglandin E 2 (PGE 2 ) and F 2α (PGF 2α ) on visfatin levels. In the present study, we have demonstrated that visfatin expression is dependent on the maturity of the animals and the stage of ovarian follicle development. Visfatin signal was detected in individual follicular compartments from primordial to antral follicles and even in atretic follicles. We have shown that the expression of visfatin in granulosa cells was higher than in theca cells. The level of visfatin is upregulated by LH, FSH, E 2, and P 4 and downregulated by insulin, while prostaglandins have modulatory effects, dependent on the dose and type of ovarian follicular cells. To summarise, our research has shown that visfatin is widely expressed in the ovarian follicles of prepubertal and mature pigs, and its expression is regulated by gonadotropins, insulin, steroids, and prostaglandins, suggesting that visfatin appears to be an important intra-ovarian factor that could regulate porcine ovarian follicular function., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Oocyte pick up (OPU) coupled with IVP produce over 1 million cattle embryos per year and has been most successful in Bos indicus derived breeds that contain large numbers of antral follicles on their ovaries. More recently, this technology has been applied on a large scale to Bos taurus cattle, where hormone manipulation is generally employed to improve the developmental competence of the COCs. Hormone manipulation, and specifically the use of FSH priming before OPU, has been strategically used in the intensively managed dairy cow, where genomic evaluation and juvenile IVP can produce additional significant genetic gains., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The objective of ovarian superstimulatory treatments in cattle is to obtain the maximum number of viable embryos by stimulating growth of antral follicles and ovulation of competent oocytes. While factors inherent to the donor animal are critical, an increased knowledge of ovarian physiology, gonadotropin biochemistry and the ability to manipulate ovarian function have provided alternatives for the design of simple and successful protocols for superovulation in cattle. Recent protocols have also been made more user-friendly and allowed for the grouping of donors for successful superovulation. Although the number of reports associating FSH profiles with superovulatory response is limited, studies designed to reduce the number of FSH treatments necessary to induce superstimulation may provide guidance for the development of optimized gonadotropin treatment protocols. Although high peak levels of circulating FSH following a single administration of Folltropin-V have been shown to be associated with a reduced superstimulatory response, the ideal treatment protocol would seem to be to increase circulating FSH levels to values comparable to those required for the induction of follicle wave emergence, and to maintain these levels for at least 72 h (or 36 h for superstimulation prior to ovum pick-up) to allow follicles to reach an ovulatory size and acquire the capacity to ovulate., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Steroid hormones are regulators in the fine-tuned process of follicular development. During final maturation in vivo a switch from oestradiol (E2) to progesterone (P4) dominance within the follicle is well-described. This change is accompanied by the resumption of meiosis and results in the maturation of the oocyte. It also suggests the important role of these hormones. However, present in vitro maturation (IVM) systems do not completely mimic the in vivo situation, resulting in oocytes of reduced quality. Aim of the study was to determine the temporal pattern of steroid hormone concentrations in the IVM medium of bovine cumulus-oocyte-complexes (COC) at defined time points. The influence of different gonadotropin supplementations during IVM on oocyte maturation, as well as the molecular quality of the oocytes and their corresponding cumulus cells was investigated. COCs were obtained from abattoir-derived ovaries and matured in medium added with different compounds of gonadotropins (eCG/hCG; FSH/LH, each at 0.05 IU or 0.01 IU; only FSH; without gonadotropins) employing a standard protocol without oil overlay. In experiment 1, medium, oocytes and cumulus cells were collected at different time points (0 h [control], 4 h, 8 h, 12 h, 16 h, 20 h, 24 h) after IVM in just eCG/hCG-supplemented medium. In experiment 2, medium, oocytes and cumulus cells were collected at 0 h (control) and after 24 h of IVM with all above-named supplements. The E2 concentration remained similar during IVM whereas P4 concentration increased during experiment 1. No significant changes could be determined after the addition of different gonadotropins (experiment 2). These results suggest that during IVM the temporal pattern of E2 and P4 did not correspond with the pattern during final maturation in vivo. RT-qPCR was used to assess the relative abundance of developmentally important genes in oocytes (BMP15; GDF9; ZAR1; PGR; PGRMC1/2; G6PD; StAR; ESR1/2; SULT1E1; STS; SOAT) and cumulus cells (ESR1/2; FSHR; LHCGR; CYP19A1; HSD3B1; PGR; PGRMC1/2; SULT1E1; STS; SOAT) at all collection points in both experiments. Most transcripts follow a time-regulated mRNA expression pattern during the entire in vitro maturation period. In addition, the expression of the analyzed transcripts was not influenced by the different gonadotropin supplementations during the IVM period. In all, this underlines that present conditions of IVM do not reflect the in vivo situation and require further optimisation., (Copyright © 2019. Published by Elsevier Inc.)

The objectives of this study were to compare three superovulation protocols and to investigate the effect of embryo diameter and shape on pregnancy rates in dromedary camel. In Experiment 1, following follicular wave synchronization of donors using two injections of GnRH (25 μg, im), superovulation was initiated 2 days after the second GnRH (Day 0 of experiment). Then, donors were assigned into three groups (n = 5) and received 1) FSH alone (390 mg), 2) eCG (1000 IU; single dose on Day 0) and FSH (330 mg) and 3) hMG (17.5 ampules). FSH and hMG were administered twice daily, in decreasing doses, for 5.5 days. Thirty-six hours after the last dose of FSH or hMG, donors were mated once. On Day 4 of superovulation, the diameter and number of follicles and on Day 7.5 after mating, total number of corpora lutea and follicles ≥9 mm were recorded using ultrasound examination. On Day 8.5 after mating, embryos were recovered non-surgically. In Experiment 2, on Day 6.5 after inducing ovulation, recipients (n = 139) received a single 7.5 days old hatched blastocyst. The diameter and shape (round, oval, collapsed) of transferred embryos were recorded. Pregnancy was detected on Day 25 after embryo transfer. Data were analyzed using Proc GLM, Proc FREG, Proc CORR and Proc Genmod of SAS. The number of corpora lutea was greater in FSH (22.4 ± 2.25) than eCG-FSH (11.6 ± 2.58) and hMG (7 ± 3.19; P < 0.01). Total number of embryos was higher in FSH (16.2 ± 2.72) than hMG (1.6 ± 1.17; P < 0.01). In FSH group, there was correlation between the ratio of follicles >6 mm/follicles ≤6 mm on Day 4 of superovulation and total number of corpora lutea (r = 0.9; P < 0.05) and embryos (r = 0.91; P < 0.05). In the latter group, there was also correlation between corpora lutea and number of embryos (r = 0.92; P < 0.05). Transfer of embryos collected from different superovulation protocols did not affect pregnancy rates (P > 0.05). The diameter of hatched blastocysts had significant effect on pregnancy rates (P < 0.05). Medium size embryos (601-985 μm) produced greater pregnancy rates (50.8%) compared to small (275-600 μm; 25%) and large (986-1571 μm; 32%) embryos (P < 0.05). In conclusion, FSH protocol provided better and predictable superovulatory response. Greater pregnancy rates could be achieved by transferring medium size hatched blastocysts in dromedary camel., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The use of oocytes recovered from prepubertal donors for in vitro embryo production has great potential for accelerating the rate of genetic gain in the dairy industry. However, these oocytes are known to be less developmentally competent than those from adult donors. In this study, we investigated the effect of age and gonadotropin stimulation in Holstein heifers subjected to oocyte collection every two weeks between 2 and 6 months of age. In order to assess the effect of gonadotropin stimulation, animals were subjected to one of three treatments, namely Short (ST; 36-42 h), Long (LT; ≥72 h) and No Treatment (NT) prior to laparoscopic ovum pick-up (LOPU). Our results show that the LT significantly improved the proportion of large follicles (>5 mm diameter) present in the ovary (LT 34.0% vs. ST 11.2% vs. NT 2.4%, P < 0.05), as well as the percentage of good-quality cumulus oocyte complexes (COCs) recovered (LT 95.3 ± 18% vs. ST 85.4 ± 22% vs. NT 82.2 ± 14%, P < 0.05) and blastocyst rate (LT 36.7 ± 26% vs. ST 18.3 ± 15% vs. NT 16.7 ± 9%, P < 0.05). Recovery rate was affected by treatment (LT 70.4 ± 25 vs. ST 85.4 ± 29 vs. NT 72.7 ± 23, P < 0.05). To assess the impact of age, data was grouped into <100 days (A), 100-130 days (B) and >130 days (C) of age at LOPU. We found that as animals got older, although the average number of COCs per donor per LOPU declined (A: 17.5 ± 11 vs. B: 14.7 ± 7 vs. C: 11.9 ± 8), the blastocyst rate increased (A: 12.8 ± 20% vs. B: 17.1 ± 21% vs. C: 21.8 ± 25%, P < 0.05). We also evaluated the incidence of polyspermy and confirmed it is a critical limitation for IVF in calf oocytes. The incidence of polyspermy was unaffected by gonadotropin treatment, but significantly decreased with age. The capacity for full development to term of in vitro produced embryos from calf oocytes was tested by embryo transfer into 21 synchronized adult recipients, which resulted in 13 pregnancies (62%), full development to term and healthy calves born. Finally, the study allowed evaluating the safety of the procedure since, on average, each animal was subjected to 8 LOPU procedures over a period of 4 months. Our results showed that the procedure is safe (no incidents during laparoscopy), and was not harmful for the reproductive future of the animals, as those that were bred became pregnant after reaching sexual maturity., (Copyright © 2017 Elsevier Inc. All rights reserved.)

A reproductive analysis of a captive group of jaguars (Panthera onca; n = 6) at the Santacruz Zoological Foundation in Cundinamarca, Colombia, was conducted by performing a longitudinal, noninvasive, hormonal analysis of estradiol and progestogens in females and of androgens in males. During four seasons, female jaguars confined in solitary were evaluated for ovarian activity and spontaneous ovulation, male jaguars for testicular activity. A second hormonal follow-up was conducted in the females after administration of gonadotropins. Hormones were extracted from fecal samples of three females (n = 3) and two males (n = 2). Estradiol measurements were obtained by RIA and progestogens by enzyme immunoassay. The linear mixed-effect regression showed that there was a significant effect of seasons in the concentrations of estradiol (chi square = 15.97, degrees of freedom = 3, P < 0.01). Posthoc comparisons of all pairs of seasonal means were conducted according to Tukey's honest significant difference, revealing significant differences between seasons: Dry 1 versus Rains 2 (P < 0.01), Rains 1 versus Rains 2 (P < 0.05), and Dry 2 versus Rains 2 (P < 0.05). Elevations of progestogens compatible with spontaneous ovulation occurred in three jaguars, and the linear mixed-effect regression showed that there was also a significant effect of seasons (chi square = 28.56, degrees of freedom = 3, P < 0.01). Posthoc comparisons showed significant differences only between seasons: Dry 2 versus Rains 2 (P < 0.01). The season with the lowest average concentration was Rains 2 (October, November, and December). During this season, periods of anestrous were registered that lasted between 31 and 83 days. The three females presented estradiol peaks after the administration of eCG. A noninvasive longitudinal analysis for androgens was also made (males 1 and 2) over the course of 1 year, and no significant differences were found between the different seasons. A seminal analysis of three adult male jaguars (Panthera onca; n = 3) was also performed after electroejaculation under general anesthesia (male 1 and 2) and by a postmortem epididymal wash (male 3). The mean concentration of spermatozoids was 5.7 × 10 6 ± 1.1 × 10 6 spermatozoa/mL. The progressive motility + standard deviation averaged 80%. The percentage of normal spermatozoids obtained by electroejaculation was 80 ± 2.8%, and the abnormalities found more frequently were head defects (7 ± 1.4%). The seminal fluid obtained by epididymal flush contained 35 ± 1.4% normal spermatozoids, and the most frequent abnormalities found corresponded to distal cytoplasmic droplets (39 ± 11.3%)., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Research was conducted to determine the effect of altrenogest and exposure to exogenous gonadotropins on ovarian function in prepubertal and mature gilts. Crossbred, presumably sexually mature gilts (n = 51), were fed with altrenogest for 18 consecutive days and the day after the last feeding with altrenogest, gilts were treated with eCG and 72 hours later challenged with hCG. Animals were slaughtered on Days 10 to 13 of their gonadotropins synchronized estrous cycle. Ovaries were examined for the number of CL, number of follicular cysts, and presence of corpora albicantia. Gilts were divided into two groups: those possessing corpora albicantia (group A-mature; n = 36) and those without corpora albicantia (Group W-prepubertal; n = 15) on their ovaries. In addition, each group was divided into two subgroups depending on the presence of follicular cysts (AC and WC) or their absence (AO and WO). There was no difference between the number of CL in group A and group W. Presence of corpora albicantia determined percentage of gilts possessing follicular cysts (13.9% group A vs. 66.7% group W). Gilts without follicular cysts (AO plus WO; n = 36) had higher number of CL (P < 0.01) than gilts bearing cysts (AC plus WC; n = 15). Comparison AO-AC did not show significant difference (P = 0.075) between CL number in mature cyst-free and cysts bearing gilts. A prepubertal gilts not bearing follicular cysts (WO) had higher (P < 0.02) number of CL than gilts bearing cysts. A significant negative correlation between the number of CL and number of follicular cysts was found (r = -0.664; P = 0.007). There were no differences in blood plasma progesterone and estradiol concentration between cyst-free and cyst-bearing gilts. These results indicate: (1) a higher follicular cysts appearance in prepubertal than mature gilts challenged with altrenogest and exposed to exogenous gonadotropins and (2) a negative effect of follicular cysts on the number of CL (ovulations) in prepubertal gilts., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Objective: The aim of this study was to clarify the correlation between clinical features (especially cyst formation and cavernous sinus invasion) and minor secretion of anterior pituitary hormones, especially adrenocorticotropic hormone (ACTH) and gonadotropin, by clinically nonfunctioning pituitary adenomas (CNFPAs) in cell culture., Methods: We examined anterior pituitary hormones secreted by samples from 63 cases of CNFPAs grown in cell culture. Magnetic resonance imaging, including determination of the Knosp grade, cystic changes, and intraoperative cavernous sinus invasion, was performed., Results: Detection of minor hormone secretion using cell culture showed that 61.9% (39/63) of CNFPAs secreted luteinizing hormone, follicle-stimulating hormone, and thyroid-stimulating hormone, 44.4% (28/63) secreted ACTH, 57.1% (36/63) secreted prolactin, and 28.6% (18/63) secreted growth hormone; only 1 case of the null-cell adenoma was observed (1.6%). Cystic changes, Knosp grade 4 status, and cavernous sinus invasion were predominantly observed in cases of ACTH-secreting adenomas. Gonadotropin-secreting adenomas showed opposite characteristics to ACTH-secreting adenomas., Conclusions: Minor secretion of ACTH and gonadotropin may be related to clinical features, especially cystic changes and invasiveness of CNFPAs, suggesting that our cell culture method could be helpful for elucidating the mechanisms of pituitary cyst formation and the invasiveness of CNFPAs., (Copyright © 2016 Elsevier Inc. All rights reserved.)

It has been previously shown that multimeric pentraxin 3 (PTX3) is a key component of the cumulus oophorus extracellular matrix (ECM) in mice. In response to the ovulatory LH surge, the cumulus cells assemble a unique ECM that envelopes the oocyte and cumulus cell complex. Importantly, cumuli from PTX3(-/-) mice were defective in their ECM organization and their fertility was impaired. It has been demonstrated that tumor necrosis factor alpha-induced protein 6 catalyzes the formation of heavy chains of (inter-alpha-trypsin inhibitor) -hyaluronan complexes and these are then cross-linked via PTX3. This process is tightly regulated and requires the proteins to meet/interact in the correct order. Finally, in this way, the above-listed proteins form the cumulus oophorus ECM. We investigated whether PTX3 is expressed in the porcine preovulatory follicle. Porcine oocyte-cumulus complexes (OCC) and mural granulosa cells (MGC) from gilts were obtained either after stimulation in vivo with eCG/hCG (4, 8, 16, 24, and 32 h) or culture in vitro (4, 24, and 44 h) in FSH/LH-supplemented medium. The methods performed were real-time reverse transcriptase-polymerase chain reaction, Western blot analysis, and immunostaining. The expression of PTX3 transcripts was significantly increased 24 h after either in vivo hCG stimulation or in vitro FSH/LH treatment in both OCC and MGC. Western blot analysis with PTX3 antibody revealed that not only matrix extracts from in vivo-stimulated gilts contain high levels of PTX3 protein but also matrix extracts of FSH/LH-stimulated OCC cultured in medium supplemented either with follicular fluid or with porcine serum. The localization of PTX3 in the cumulus oocyte complex was confirmed by immunostaining. In conclusion, PTX3 is produced by porcine OCC and MGC both in vivo and in vitro with gonadotropin stimuli inducing cumulus expansion., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Maintaining follicle integrity during development, whereby each follicle is a functional unit containing a single oocyte, is essential for the generation of healthy oocytes. However, the mechanisms that regulate this critical function have not been determined. In this paper we investigate the role of the oocyte in maintaining follicle development. To investigate this role, we use a mouse model with oocyte-specific deletion of C1galt1 which is required for the generation of core 1-derived O-glycans. The loss of oocyte-generated O-glycans results in the joining of follicles and the generation of Multiple-Oocyte Follicles (MOFs). The aim was to determine how Mutant follicle development is modified thus enabling follicles to join. Extracellular matrix and follicle permeability were studied using histology, immunohistochemistry and electron microscopy (EM). In ovaries containing Mutant Oocytes, the Follicle basal lamina (FBL) is altered both functionally and structurally from the primary stage onwards with Mutant follicles possessing unexpectedly thicker FBL. In Mutant ovaries, the theca cell layer is also modified with intermingling of theca between adjacent follicles. MOF function was analysed but despite increased numbers of preantral MOFs in Mutants, these do not reach the preovulatory stage after gonadotrophin stimulation. We propose a model describing how oocyte initiated changes in FBL and theca cells result in follicles joining. These data reveal new and important roles for the oocyte in follicle development and follicle integrity., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Follicular fluid meiosis-activating sterol (FF-MAS), an intermediate in the cholesterol biosynthetic pathway, has been identified as a compound that induces the resumption of meiosis in mammalian oocyte. Follicular fluid meiosis-activating sterol is converted to testis meiosis-activating sterol by a sterol Δ14-reductase. An inhibitor of Δ14-reductase and Δ7-reductase, AY9944 A-7, causes accumulation of FF-MAS by inhibiting its metabolism. The objective of this research was to investigate the specific contribution of AY9944 A-7 on gonadotropin-induced meiotic resumption and its interactive effects with FSH or LH on meiotic maturation of oocytes and preimplantation development of parthenogenetic embryo in sheep by addition of AY9944 A-7 during IVM to cause accumulation of FF-MAS. First, ovine cumulus-oocyte complexes (COCs) were cultured in the presence of FSH (10 μg/mL), LH (10 μg/mL), AY9944 A-7 (20 μmol/L), FSH (10 μg/mL)+AY9944 A-7 (20 μmol/L), or LH (10 μg/ml) + AY9944 A-7 (20 μmol/L) with an inhibitor hypoxanthine (Hx) to prevent spontaneous meiosis of oocytes. The resumption of meiosis was assessed by the frequency of germinal vesicle breakdown and the first polar body (PBI) extrusion. The kinetics of gonadotropin and AY9944 A-7-induced meiotic resumption in vitro was also evaluated in the study. The numbers of oocytes resuming meiosis and undergoing germinal vesicle breakdown were counted after the COCs were cultured for 2, 4, 8, 12, 16, 20, and 24 hours. Matured oocytes extruding PBI were selected for parthenogenetic activation, and the percentages developing to the two-cell stage and blastocyst stage were recorded as indicators of parthenogenetic embryo developmental competence. It was observed that FSH could induce the resumption of meiosis of ovine COCs cultured in the presence of Hx, but LH could not. AY9944 A-7 had a synergistic effect with FSH on nuclear maturation and developmental competence of embryos produced by parthenogenetic activation, whereas it had no added advantage on LH action. However, the kinetics of meiotic resumption after AY9944 A-7 stimulation was remarkably delayed when compared with FSH-induced maturation. In conclusion, the current study suggested that AY9944 A-7 supplementation in IVM medium optimized the beneficial effects of FSH on meiotic maturation of ovine oocytes and subsequent developmental competence of embryos produced by parthenogenetic activation. This work had important potential for developing a novel technique in IVM of ovine oocytes., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Ovarian stimulation is used with IVF/intracytoplasmic sperm injection (ICSI) cycles to obtain multiple oocytes and improve pregnancy rates; however, it also induces perturbation in the oxidant-antioxidant balance leading to oxidation stress. The present study monitored the plasma antioxidant status in women undergoing a long agonist protocol of ovarian stimulation at three different time points: at baseline (T0), after pituitary suppression (T1) and on the day of oocyte retrieval (T2). The antioxidant composition of follicular fluid samples collected on T2 was also evaluated. Significant decreases (P < 0.05) of plasma vitamin C, vitamin E and carotenoids were found between T1 and T2 but not between T0 and T1. At T2, high plasma vitamin E was associated with high numbers of total and mature oocytes retrieved per patient, which, in turn, were favourable for achieving pregnancy. Accordingly, women who became pregnant presented higher vitamin E concentrations both in plasma and FF than those who did not. In conclusion, this study confirmed the occurrence of significant modifications of the plasma antioxidant profile during ovarian stimulation with gonadotrophins; at the same time, it was found that both systemic and follicular antioxidant status may be related to IVF/ICSI outcome., (Copyright © 2014 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.)

Prostaglandin E2 (PGE2) is an autocrine/paracrine factor which mediates gonadotrophin (Gn) stimulation of cumulus expansion and oocyte maturation in rodents. Its role in bovine oocyte maturation is less characterized. This study detected PTGS2 (COX2) and PGE synthases (PTGES1, PTGES2 and PTGES3) in bovine cumulus-oocyte complexes (COC). Only PTGS2 and PTGES1 expression changed during maturation. In Gn-free media, no cumulus expansion and ∼45% nuclear maturation was achieved, while Gn-induced maturation showed full cumulus expansion (score 3) and ∼87% maturation. PGE2 supplementation without Gn induced mild cumulus expansion (score 0.5-1) but increased nuclear maturation to levels similar to those obtained with Gn alone. In the presence of Gn, exogenous PGE2 did not affect expansion or nuclear maturation and subsequent embryo development. Treatment with PTGS2 selective inhibitor (NS398), PTGS2-specific siRNA or PTGER2-receptor antagonist (AH6809) resulted in ∼20-25% reduction in nuclear maturation. NS398 and AH6809 did not affect cumulus expansion. Most oocytes not reaching metaphase of second meiosis (MII) following NS398, AH6809 and PTGS2-specific siRNA treatments were at MI. After longer maturation, NS398-treated oocytes had normal MII rate and uncompromised embryo development. PGE2 has a limited role in cumulus expansion in bovine COC but is important for the timing of Gn-induced nuclear maturation. We confirmed that genes involved in the synthesis of prostaglandin E2 (PGE2) are expressed by cumulus-oocyte complexes (or eggs) of cows and that PGE2 is synthesized during oocyte maturation in the presence of gonadotrophin hormones. When we inhibited synthesis of PGE2 or blocked its receptors, oocyte maturation, but not cumulus expansion, was compromised. Further investigation showed that oocyte maturation is delayed but not arrested when PGE2 synthesis is inhibited. On the other hand, addition of exogenous PGE2 induced a high maturation rate and mild cumulus expansion only in the absence of gonadotrophin stimulation, and had no effect in the presence of gonadotrophin., (Copyright © 2013 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.)

This study investigated the factors associated with utilization of fertility preservation and the differences in treatments and outcomes by prior chemotherapy exposure in patients with haematological diseases. This study included all 67 women with haematological diseases seen for fertility preservation consultation at two university hospitals between 2006 and 2011. Of the total, 49% had lymphoma, 33% had leukaemia, 7% had myelodysplastic syndrome and 4% had aplastic anaemia; 46% had prior chemotherapy; and 33% were planning for bone marrow transplantation, 33% pursued ovarian stimulation and 7% used ovarian tissue banking; and 48% of patients did not pursue fertility preservation treatment. All five cycle cancellations were in the post-chemotherapy group: three patients with leukaemia and two with lymphoma. Patients with prior chemotherapy had lower baseline antral follicle count (10 versus 22) and received more gonadotrophins to achieve similar peak oestradiol concentrations, with no difference in oocyte yield (10.5 versus 10) after adjustment for age. Embryo yield was similar between those who had prior chemotherapy and those who had not. Half of the patients with haematological diseases who present for fertility preservation have been exposed to chemotherapy. While ovarian reserve is likely impaired in this group, oocyte yield may be acceptable., (Copyright © 2013 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.)

Study Objective: To evaluate the effects of salpingectomy on the ovarian response to gonadotropins and in vitro fertilization-embryo transfer (IVF-ET) cycle outcomes in women with tubal factor infertility., Design: A retrospective study (Canadian Task Force Classification II-3) SETTING: An in vitro fertilization laboratory in a university hospital in Taiwan., Patients: We analyzed the outcomes of 288 consecutive fresh IVF-ET cycles in 251 consecutive women with tubal factor infertility from January 2001 to December 2011. Two hundred eighty-eight cycles were divided into 2 groups comprising 103 cycles with laparoscopic salpingectomy and 185 cycles with prior bilateral tubal sterilization, laparoscopic tuboplasty, or proximal tubal occlusion as the control group., Interventions: Controlled ovarian hyperstimulation and IVF-ET., Measurements and Main Results: The main outcome was measured by comparing the duration of stimulation, number of gonadotropin ampoules per cycle, number of follicles, number of oocytes retrieved, fertilization rate, implantation rate, clinical pregnancy rate, and live birth rate. We observed no significant difference in any ovarian response parameter between the salpingectomy and nonsalpingectomy groups. Implantation rates, clinical pregnancy rates, and live birth rates were similar. The mean numbers of follicles and oocytes retrieved ipsilateral to the operated side in the salpingectomy group were similar to the numbers of follicles and oocytes retrieved from the nonoperated ovary., Conclusions: Laparoscopic salpingectomy did not have a negative effect on the ovarian response in women with tubal factor infertility., (Copyright © 2013 AAGL. Published by Elsevier Inc. All rights reserved.)

The ability to predict the response potential of women to ovarian stimulation may allow the development of individualized ovarian stimulation protocols. This tailored approach to ovarian stimulation could reduce the incidence of ovarian hyperstimulation syndrome in women predicted to have an excessive response to stimulation or could improve pregnancy outcomes in women classed as poor responders. Namely, variation of the type of gonadotrophin-releasing hormone (GnRH) analogue or the form and dosage of gonadotrophin used for stimulation could be adjusted according to an individual's response potential. The serum concentration of anti-Müllerian hormone (AMH) is established as a reliable marker of ovarian reserve, with decreasing concentrations correlated with reduced response potential. This review examines the current evidence evaluating individualized ovarian stimulation protocols using AMH concentration as a predictive marker of ovarian response. The rationale behind why specific treatment protocols based on individual response potential may be more suitable is also discussed. Based on current evidence, it appears that the use of AMH serum concentrations to predict ovarian response and optimize treatment strategies is a promising approach for improving pregnancy outcomes in women undergoing ovarian stimulation. However, prospective randomized controlled trials evaluating this approach are needed before any firm conclusions can be drawn., (Copyright © 2013 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.)

Thyroxine (T4) plus gonadotropins might stimulate ovarian follicular angiogenesis in immature infertile hypothyroid rdw rats by upregulating mRNA expression of major angiogenic factors. Development of growing corpus luteum (CL) is strongly related to angiogenesis and to morphofunctional development of microcirculation. Our aim was to investigate if T4 is involved in CL angiogenesis and in the activation of capillary cells and angiogenic factors after ovulation in a spontaneous model of hypothyroidism, the rdw rat. Rdw rats were treated with T4 plus gonadotropins (equine chorionic gonadotropin plus human chorionic gonadotropin; eCG+hCG) or gonadotropins alone in order to evaluate the effects of T4 on early luteal angiogenesis, on microvascular cells and on expression of major growth factors which are involved in the regulation of angiogenesis. Wistar-Imamichi rats treated with gonadotropins were used as controls. The ovaries were collected 4 days after hCG administration and analyzed using morphologic and molecular approaches. Thyroxine plus gonadotropins stimulated the growth of CLs and follicles as in controls, differently from rdw rats treated only with gonadotropins, in which CLs were not found and only small follicles, often atretic, could be recognized. In T4 plus gonadotropin-treated rdw rats CLs showed increased microvasculature, numerous activated capillaries characterized by sprouting and other angiogenic figures, and associated pericytes. Quantitative analysis revealed that the number of pericytes in T4 plus gonadotropin-treated rdw rats was comparable with that found in control rats and was significantly higher than that found in gonadotropin-treated rdw rats. The mRNA expression of vascular endothelial growth factor and basic fibroblast growth factor was significantly higher in control rats and in T4 plus gonadotropin-treated rdw rats than in gonadotropin-treated rdw rats. mRNA expression of tumor necrosis factor α, transforming growth factor β, and epidermal growth factor did not show significant changes. Our data originally demonstrated that T4 promoted the growth of an active microcirculation in developing CLs of gonadotropin-primed hypothyroid rdw rats, mainly by inducing sprouting angiogenesis, pericyte recruitment, and upregulation of mRNA expression of vascular endothelial growth factor and basic fibroblast growth factor. In conclusion, we suggest that T4 plays a key role in restoring luteal angiogenesis in ovaries of immature hypothyroid rdw rats., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Objective: To evaluate by the birth rate the impact of the number of days of estrogens continued beyond the menses in a four days estradiol IVF antagonist programming cycles., Patients and Methods: Retrospective study from September 2004 to January 2009 among women of age ranging between 25 and 38 years. Four milligrams of provames is prescribed 3 to 5 days before the theorical menses and continued until the beginning day of stimulation, which is distributed equitably between Thursday and Sunday. The birth rate is evaluated according to the number of days of estrogen continued beyond the menses within a limit from 1 to 8., Results: No significant difference appears neither in the duration of stimulation, in the quantity of gonadotrophin, the oocytes pick up, nor in the rate of birth between the groups., Conclusion: The programming by estrogens of the antagonist IVF cycles implies a variable number of days of estrogens continued beyond the menses, which does not seem to affect the birth rate., (Copyright © 2011 Elsevier Masson SAS. All rights reserved.)

Tumor necrosis factor α (TNFα) acts through two receptors, TNFα receptor| (TNFR|) and TNFα‖ (TNFR‖). Tumor necrosis factor α receptor| knockout mice had early senescence and poor fertility, whereas TNFR‖ knockout mice had reproductive performance not different from wild type (WT) mice. In the present study, TNFα knockout mice were used to study the roles of TNFα in female reproduction. The TNFα-/- mice had similar vaginal opening time (PD 27.6 ± 1.8 vs PD 27.7 ± 1.9, respectively, P > 0.05) and exogenous gonadotropin primed TNFα-/- mice shed more ova (28.9 ± 3.75 vs 9.8 ± 0.51, respectively, P = 0.001) compared with WT controls. At 2 mo of age, in 21 d, TNFα-/- mice had more estrous cycles than WT counterparts (6.0 ± 0.25 vs 4.0 ± 0.28, respectively, P < 0.05). Tumor necrosis factor α mutation also influenced ovarian follicular development; TNFα-/- mice had approximately a two-fold larger follicle pool in the early neonatal period (6087 ± 508.15 vs 3440 ± 261.91, respectively, P = 0.004), whereas TNFα knockout affected growth of primordial follicles to the antral stage as well. Moreover, TNFα-/- mice gave birth to 21% more pups than control mice during a 12 mo breeding period (37.38 ± 3.69 vs 22.38 ± 3.53, respectively, P = 0.03). At 1 y of age, the follicular reserve in TNFα-/- mice was more than that in WT mice. These physiological differences in TNFα-/- mice were associated with increased proliferation of granulosa cells and decreased apoptosis of oocytes. This was apparently the first demonstration that in the TNFα-/- mouse model, multiple parameters of ovarian function were altered, and that lack of TNFα increased fertility in mice., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Hypogonadotrophic hypogonadism (HH) is characterized by deficient gonadotrophin secretion, resulting from pituitary or hypothalamic defects. In order to induce spermatogenesis, HH patients are treated with commercially available gonadotrophins. As far as is known, quality and genetic integrity of induced sperm cells have never been investigated, although they represent an important issue, since the ultimate goal of this treatment is to have competent spermatozoa in order to achieve paternity. In order to evaluate the nuclear integrity of induced sperm cells, sperm samples from treated HH patients were compared with sperm samples from normospermic control donors. Sperm cells were analysed by fluorescence in-situ hybridization, using probes specific for chromosomes 13, 21, 18, X and Y, and by TdT (terminal deoxynucleotidyl transferase)-mediated dUDP nick-end labelling assay. Results showed that the rate of aneuploid and diploid sperm cells in patients was not statistically different from controls and that the rate of sperm cells with fragmented DNA was within the normal values. Spermatozoa obtained by gonadotrophin treatment in HH patients are likely to have a balanced chromosomal content and a normal DNA integrity but this conclusion needs to be confirmed by further studies dealing with a greater number of patients., (Copyright © 2010 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.)

During luteinization, circulating high-density lipoproteins supply cholesterol to ovarian cells via the scavenger receptor-B1 (SCARB1). In the mouse, SCARB1 is expressed in cytoplasm and periphery of theca, granulosa, and cumulus cells of developing follicles and increases dramatically during formation of corpora lutea. Blockade of ovulation in mice with meloxicam, a prostaglandin synthase-2 inhibitor, resulted in follicles with oocytes entrapped in unexpanded cumulus complexes and with granulosa cells with luteinized morphology and expressing SCARB1 characteristic of luteinization. Mice bearing null mutation of the Scarb1 gene (SCARB1(-/-)) had ovaries with small corpora lutea, large follicles with hypertrophied theca cells, and follicular cysts with blood-filled cavities. Plasma progesterone concentrations were decreased 50% in mice with Scarb1 gene disruption. When SCARB1(-/-) mice were treated with a combination of mevinolin [an inhibitor of 3-hydroxy-3-methylglutaryl CoA reductase (HMGR)] and chloroquine (an inhibitor of lysosomal processing of low-density lipoproteins), serum progesterone was further reduced. HMGR protein expression increased in SCARB1(-/-) mice, independent of treatment. It was concluded that theca, granulosa, and cumulus cells express SCARB1 during follicle development, but maximum expression depends on luteinization. Knockout of SCARB1(-/-) leads to ovarian pathology and suboptimal luteal steroidogenesis. Therefore, SCARB1 expression is essential for maintaining normal ovarian cholesterol homeostasis and luteal steroid synthesis.

Ovarian carcinoma is the leading cause of death among gynecologic cancers. Although transformation of the outer ovarian epithelium was linked with ovulation, the disease is significantly more prevalent and severe in postmenopausal women. We postulated that menopause could augment ovarian cancer progression through the effects of gonadotropins on multifocal seeding to the mesothelial layer lining the peritoneum. This seeding is mediated by integrins as well as by CD44 interaction with hyaluronan (HA). Here, we report the effect of gonadotropins on HA synthesis and degradation and on peritoneal adhesion. A significant concentration- and time-dependent induction in expression levels of HA synthases (HASs) and hyaluronidases (Hyals) was observed in vitro on stimulation of human epithelial ovarian carcinoma cells by gonadotropins. Hormonal regulation of HA-mediated adhesion was manifested in vivo as well, by fluorescence microscopy of stained MLS multicellular tumor spheroids. The number of spheroids adhered to the mesothelium of ovariectomized CD-1 nude mice 9.5 hours after intraperitoneal insertion was significantly higher than in nonovariectomized mice. Inhibition of HA synthesis by 6-diazo-5-oxo-1-norleucine (DON) both in spheroids and ovariectomized mice significantly reduced the number of adhered spheroids. Thus, the change in the hormonal environment during menopause assists in HA-dependent adherence of ovarian cancer spheroids onto the peritoneum. However, HA is antiangiogenic and it can significantly suppress tumor progression. Accordingly, angiogenesis of the adhered spheroids was significantly elevated in DON-treated tumors. These results can explain the selective pressure that can lead to simultaneously increased tumor expression of both HASs and Hyals.

This study aimed first to evaluate the effect of recombinant human FSH (rhFSH) with and without recombinant human LH (rhLH) on fresh and frozen-thawed embryo development and also to analyze the immune response of rabbit does (Oryctolagus cuniculus) subjected to repeated rhFSH treatments. Nulliparous New Zealand White does were used. In Experiment 1, 120 does were superovulated with 25 IU rhFSH alone or in combination with 5% or 10% rhLH (1.25 IU or 2.50 IU rhLH). A total of 1116 embryos at the compacted morula stage were cultured at 38.5 degrees C, 5% CO(2), and saturated humidity for 48 h. The embryo development to hatching blastocyst was significantly lower for the group with 10% rhLH versus that of the control group (65.6 vs. 79.5 for rhFSH+10% rhLH vs. control, respectively). However, no significant difference was found in development to hatching blastocyst for the control, rhFSH alone, and rhFSH+5% rhLH groups. The developmental potential of frozen-thawed embryos obtained from all groups was similar, with an 83.5% in vitro development rate until the expanded blastocyst stage. To detect anti-FSH antibodies, in Experiment 2, does were subject to four superovulation treatments. The hormone administration had a significant effect on immune response in the superovulation group after two treatments (0.14+/-0.074 and 0.15+/-0.076 vs. 0.46+/-0.078 and 0.50+/-0.078 optical density for the first, second, third, and forth cycles, respectively). Nevertheless, none of the treated does had an immune response in both the first and second treatments; on the contrary, a significant increase in the antibody levels was observed in these females at the moment of the third and fourth superovulation treatments. In conclusion, rhFSH superovulation treatments increase the reproductive potential of rabbit does.

The objective was to study dynamic changes of ovaries in rhesus macaques stimulated by gonadotropins to identify an indicator for predicting ovarian response to stimulation. Twenty-one cycling monkeys were given 36 IU/d recombinant human follicle-stimulating hormone (rhFSH) for 8 d. Animals (n=17) with > or =5 follicles (> or =3mm) in their ovaries on Day 9 of ovarian stimulation were deemed good responders, whereas those with a lesser response were poor responders (n=4). For these two groups, the mean (+/-SD) numbers of oocytes retrieved were 44.3+/-21.4 and 11.0+/-4.6, respectively. In retrospect, the mean diameters of the ovaries and of the largest follicles, the total number of detectable follicles (diameter >0.5mm), and serum estradiol concentrations gradually increased during the stimulation period in the good responders but did not increase in the poor responders. Comparing good and poor responders, the number of ovarian follicles >0.5mm already exhibited a difference (12.9+/-6.5 vs. 2.9+/-1.3, respectively, P<0.05) on Day 1 of stimulation. However, for other end points, differences were not significant until at least Day 5. Moreover, good responders yielded a fivefold higher blastocyst development rate than that of poor responders (P<0.01). In conclusion, the number of ovarian follicles detected with ultrasonography could be useful to predict the response to FSH stimulation in non-human primates.

Similarities in the phenotype observed in women with FSH receptor mutation and in FSH receptor knockout mice have clearly established a critical role of this protein in normal gonadal function. Two common single nucleotide polymorphisms in the exonic region of the FSH receptor gene have been shown to be associated with altered ovarian response in subjects undergoing gonadotrophin treatment. Recent in-vitro studies have shown that the A allele at the -29 position in the 5 untranslated region of the FSH receptor gene is associated with impaired transcriptional activity. Differential expression of the FSH receptor and its function may be one of the factors responsible for altered ovarian response. These observations prompted a study of the association between FSH receptor genotype at the -29 position and ovarian response in women undergoing gonadotrophin treatment. Analysis of the data revealed that the subjects with AA genotype at the -29 position required the highest amount of exogenous FSH for ovulation induction, and oestradiol concentrations before the day of human chorionic gonadotrophin administration were significantly lower (P = 0.015) compared with the GA genotype. The number of pre-ovulatory follicles and retrieved oocytes were lowest in the subjects with AA genotype. These results indicate that the AA genotype at position -29 may be associated with the poor ovarian response.

Hypogonadotrophic hypogonadism is characterized by oligospermia or azoospermia and low testosterone, FSH and LH concentrations. In such cases, treatment with gonadotrophins is required to produce or increase spermatozoa in the ejaculate but few achieve normal spermatogenesis. After long periods of medical treatment, if the patients still have a low sperm count or azoospermia, assisted reproductive technologies in addition to hormone administration can be offered. Four cases of hypogonadotrophic hypogonadism with persistent azoospermia after at least 10 months of medical treatment are reported. In all four cases, spermatozoa retrieved by testicular sperm extraction and intracytoplasmic sperm injection (ICSI) were used to achieve fertilization. Excess spermatozoa were frozen in all cases. Six ICSI cycles using fresh testicular spermatozoa in four and thawed testicular spermatozoa in two were performed. Although there was no pregnancy in cycles where thawed spermatozoa were used, three clinical pregnancies were achieved in four cycles using fresh testicular spermatozoa. One of them ended with spontaneous abortion at 10 weeks of gestation and the two others resulted in the delivery of three normal offspring. If azoospermia persists after medical treatment, spermatozoa can be obtained surgically from testes and can be used successfully to achieve pregnancy in cases of hypogonadotrophic hypogonadism.

Despite the probable inhibitory effects of GnRH analogues on ovarian steroidogenesis in vitro, their association with assisted reproduction protocols shows favorable results. This suggests that there are important differences in the behaviors of these drugs when administered in vivo versus in vitro. To clarify these differences, this study was designed to analyze the effect of leuprolide acetate (LA) on ovarian steroidogenesis in women undergoing In Vitro Fertilization (IVF). A prospective, randomized open label study was conducted on 14 women (26-35 years): seven receiving only gonadotrophins (Group 1) and seven receiving gonadotrophin plus LA at 1mg/day (Group 2). The LA in vivo effect was determined with serum and follicular fluid (FF) samples and via luteinized granulosa cell cultivation (GCC), where cells were obtained during oocyte retrieval after ovarian hyperstimulation. In vitro analysis was performed via addition of LA to GCC only for Group 1 (without LA) at progressively higher concentrations (0, 10(-12), 10(-9) and 10(-6)M). In vivo, the main observation was a reduction in androgen production in Group 2, represented by lower androstenedione production in FF (G1=6479+/-3458; G2=3021+/-1119 ng/ml; p=0.04) and a lower testosterone peak in GC at 96h (G1=0.64+/-0.12 ng/ml; G2=0.50+/-0.19 ng/ml; P=0.02), but a higher fertilization rate (G1=67%; G2=83%; p=0.009). In vitro, testosterone, estradiol and progesterone were also reduced by LA, even though this reduction occurred for progesterone only at the highest LA dosage (10(-6)M; 606.0+/-114.3 ng/ml versus 1524.0+/-246.5 ng/ml; p=0.02). Results show that LA reduces ovarian steroidogenesis in vivo by essentially inhibiting androgen synthesis; whereas, in vitro, ovarian steroidogenesis is reduced overall.

Female mice null for receptor-interacting protein 140 (RIP140) are infertile because of the failure of follicle rupture. The present study examined gonadotropin regulation of RIP140 expression in immature rat ovary. Treatment with PMSG increased ovarian RIP140 mRNA and protein levels. In contrast, hCG treatment rapidly inhibited RIP140 mRNA and protein levels within 1-3 h. RIP140 mRNA was detected in theca cells of growing follicles in untreated ovary and in granulosa cells in PMSG-treated ovary. Interestingly, hCG treatment reduced RIP140 mRNA levels in granulosa cells of preovulatory follicles, but not of growing follicles. Neither treatment of immature rats with diethylstilbestrol in vivo nor of immature granulosa cells with FSH in vitro affected RIP140 mRNA levels. Treatment of immature granulosa cells with 17beta-estradiol in vitro, however, stimulated RIP140 mRNA levels. In cultured preovulatory granulosa cells, RIP140 mRNA levels were stimulated at 1 h and then declined to below control levels by 3 h after LH treatment. Treatment with MDL-12,330A, an inhibitor of adenylate cyclase, or chelerythrine chloride, an inhibitor of protein kinase C (PKC), inhibited LH-stimulated RIP140 gene expression. Furthermore, forskolin or TPA treatment for 1 h mimicked the stimulatory action of LH, indicating the involvement of both adenylate cyclase and PKC pathways. These results demonstrate the stimulation by PMSG and inhibition by hCG of RIP140 expression in granulosa cells of preovulatory follicles in the rat ovary.

Objective: This study determines whether pretreatment levels of müllerian inhibiting substance/antimüllerian hormone (MIS/AMH) would reflect ovarian response to exogenous gonadotropin in women with polycystic ovary syndrome (PCOS) and ovulatory controls matched by age and weight., Study Design: Case-control study of 20 women with PCOS and 10 normoovulatory women undergoing controlled ovarian hyperstimulation (COH) for in vitro fertilization (IVF) at an academic medical center., Results: Baseline serum MIS/AMH levels in PCOS were higher than those of normoovulatory women (P < .001). MIS/AMH levels increased after gonadotropin-releasing hormone (GnRH) agonist pituitary suppression; 0.5 ng/mL in PCOS (P = .12) and 0.7 ng/mL in controls (P < .02). In normoovulatory women, MIS/AMH at baseline, after pituitary suppression, and the interval change after pituitary suppression all correlated closely to the number of mature oocytes retrieved (P < .005). In PCOS, however, levels of MIS/AMH at baseline and after pituitary suppression did not show this correlation, whereas only the interval change correlated with the number of mature oocytes retrieved., Conclusion: Baseline MIS/AMH is a good predictor of the ovarian response to COH in normoovulatory women but not in PCOS.

The expression of lanosterol 14alpha-demethylase (LDM) in the mouse ovary after gonadotrophin administration was examined and the action of follicle fluid meiosis activating sterol (FF-MAS), derived from lanosterol by the action of LDM, on oocyte spontaneous maturation was also evaluated in cumulus cell enclosed oocytes (CEOs). Expression of LDM was primarily in oocytes in primordial and secondary follicles prior to administration of gonadotrophins, but obvious LDM expression was apparent in ovarian somatic cells 48 h after administration of equine chorionic gonadotrophin (eCG), especially in luteal and cumulus cells 54 h after eCG or 48 h after eCG plus 6 h after human chorionic gonadotrophin (hCG). The LDM expression in oocytes was only slightly elevated in larger growing follicles after eCG treatment. On the contrary, 48 h after hCG treatment, the elevated expression of LDM was only detected in interstitial cells. Therefore, eCG may be the primary gonadotrophin for LDM expression, and furthermore for production of FF-MAS in mouse cumulus cells (which are indispensable for oocyte maturation in vivo). Conversely, inhibitors of LDM, either 40 microM azalanstat or 50 microM RS-21745, significantly inhibited oocyte germinal vesicle breakdown (GVB) after 4h of in vitro culture; GVB rates decreased to 14 or 20%, compared to 90% in spontaneous maturation, respectively. There was no significant increase in GVB in CEOs following specific inhibitor of sterol Delta14-reductase and Delta7-reductase, AY9944-A-7 (5-100 microM), until marked oocytes degeneration appeared (50 microM). The phenomena may be ascribed to slow, passive accumulation of FF-MAS by AY9944-A-7, which cannot be associated with fast spontaneous progression. Furthermore, in spontaneous-matured CEOs, LDM was expressed preferentially in cumulus cells instead of oocytes. Therefore, FF-MAS may have a positive role in the spontaneous maturation of CEOs. In conclusion, there was an eCG-dependent dual LDM expression pattern on both oocytes and somatic cells in growing follicles in vivo, which may increase LDM expression and FF-MAS production in cumulus cells for oocyte maturation. For the first time, the inhibitory effect of LDM inhibitors on spontaneous maturation, together with the strong LDM expression in spontaneous matured CEOs, indicated that FF-MAS produced by cumulus cells might participate in spontaneous maturation of mouse CEOs.

Embryo transfer practice and results were examined over a 20-year period in Holstein cows and heifers within four commercial embryo transfer programs located in different areas of North America. Mean embryo production per collection decreased (P < 0.05) in one program over time, but not in the other three. Changes in the type of cows entering embryo transfer programs, the number of times they were superstimulated and changes in the brands of gonadotropins used for superstimulation all complicated the analysis of embryo production over time. Data reveal higher pregnancy rates (P < 0.001) following transfer of embryos into Holstein heifers than into lactating dairy cows. It is not clear whether pregnancy rates have decreased over time as a result of the change from surgical to non-surgical embryo transfer. In the two programs in which pregnancy rates were analyzed, there was a decrease (P < 0.001) when non-surgical transfers were adopted in one program, while no change occurred in the other. One of the biggest changes in all programs was that more than 50% of embryos recovered from donors are now frozen after collection, whereas the majority were transferred fresh 20 years ago.

Due to inherent differences between gonadotrophin-releasing hormone (GnRH) antagonists and agonists, their late effect on ovarian steroidal production during the luteal phase of IVF cycles may differ. The aim of this study was to characterize and compare the luteal phase hormonal profile after the use of GnRH antagonists or agonists in ovarian stimulation protocols for IVF, in non-conception cycles, to avoid the effect of human chorionic gonadotrophin (HCG) during the luteal phase in conception cycles. Seventy-eight normo-ovulatory patients <35 years old, undergoing IVF due to male or tubal infertility were randomly allocated either to a GnRH antagonist (study group) or GnRH agonist treatment (control group). Similar standard luteal support was given to all patients, using vaginal micronized progesterone. In non-conception cycles, no statistically significant differences were found comparing luteal phase. oestradiol or progesterone levels in the study and control groups. No statistically significant differences were found comparing the hormonal profile dynamics, the mid-luteal (HCG day +8) oestradiol/progesterone ratio and the percentage of mid-luteal oestradiol decline between the study and control groups. In conclusion, similar characteristics and dynamics of luteal phase oestradiol and progesterone were demonstrated comparing ovarian stimulation for IVF using GnRH agonist or antagonists, under similar luteal support.

The current study aimed to investigate whether single day-3 embryo transfer (SET) results in higher early pregnancy loss (EPL) than single blastocyst transfer (SBET). A total of 896 patients underwent 1103 IVF cycles with a gonadotrophin-releasing hormone (GnRH) antagonist protocol. In 603 cycles (D3 group) a single embryo on day 3 of the embryo culture was transferred, whereas in the remaining 500 cycles a single blastocyst was transferred on day 5 (D5 group). Multifollicular ovarian stimulation was performed with a GnRH antagonist protocol starting on day 6. SET resulted in 209 pregnancies (34.7%), compared with 221 pregnancies (44.2%) for SBET. Early pregnancy loss rate was significantly higher with SET compared with SBET (26.8% versus 17.2%, P = 0.017) and ongoing implantation rate was also significantly higher with day 5 compared with day-3 embryo transfer (OR:1.68, 95% confidence interval:1.31-2.18). Sub-optimal embryo selection for transfer on day 3, in addition to asynchronization between altered endometrium and early exposure of cleavage-stage embryos, might explain the above difference. Nevertheless, the higher implantation potential of the blastocyst questions the rationale behind performing single embryo transfer on day 3 of embryo culture in women under 36 years old.

Progesterone acts at a plasma membrane receptor on the Rana oocyte to initiate meiosis. A cascade of lipid messengers occurs within seconds, followed by sequential changes in membrane phospholipid composition. We now show that progesterone binding to the plasma membrane increases continuously over the first 4 h. Subsequently, about 60% of the total plasma membrane and > 90% of membrane-bound progesterone, ouabain binding sites, and Na/K-ATPase activity are internalized. Until the completion of membrane internalization, oocytes must be continuously exposed to nanomolar concentrations of exogenous progesterone for meiosis to continue. The membrane-bound progesterone remains unchanged, whereas microinjected [(3)H]progesterone is rapidly metabolized. We find that progesterone and the plant steroid ouabain compete for one of two ouabain binding sites on the oocyte surface. Ouabain blocks progesterone action and inhibits subsequent meiosis if added at any time during the first 4-5 h. Western blots of SDS/PAGE extracts of isolated oocyte plasma membranes contain a -110 kDa band which binds an antibody to the steroid-binding c-terminal domain in rat and human PR. The number of binding sites and K(d) for progesterone binding to the plasma membrane is comparable to those for low-affinity ouabain binding to the alpha-subunit of the Na/K-ATPase (112 kDa). Our results suggest that progesterone binding to the ouabain binding site on the N-terminal region of the alpha-subunit of Na/K-ATPase may modulate early plasma membrane events over the first 4-6 h. Progesterone may thus act in part through the plasma membrane Na/K-ATPase signaling system.

The molecular basis for acquisition of meiotic and developmental competence, the two main outcomes of oocyte development and essential for producing an egg capable of being fertilized and supporting development to term, is largely unknown. Using microarrays, we characterized global changes in gene expression in oocytes derived from primordial, primary, secondary, small antral, and large antral follicles and used Expression Analysis Systematic Explorer (EASE) to identify biological and molecular processes that accompany these transitions and likely underpin acquisition of meiotic and developmental competence. The greatest degree of change in gene expression occurs during the primordial to primary follicle transition. Of particular interest is that specific chromosomes display significant changes in their overall transcriptional activity and that in some cases these changes are largely confined to specific regions on these chromosomes. We also examined the transcript profile of oocytes that developed in vitro, as well as following eCG priming. Remarkably, the expression profiles only differed by 4% and 2% from oocytes that developed in vivo when compared to oocytes that developed in vitro from either primordial or secondary follicles, respectively. About 1% of the genes were commonly mis-expressed, and EASE analysis revealed there is an over-representation of genes involved in transcription. Developmental competence of oocytes obtained from eCG-primed mice was substantially improved when compared to oocytes obtained from unprimed mice, and this correlated with decreased expression of genes implicated in basal transcription.

Clomiphene (clomiphene citrate) remains the drug of first choice in women suffering from anovulation and in ovulating women with inadequate follicular or luteal development. In ovarian stimulation for various assisted reproductive techniques, the role of clomiphene remains limited, because the use of gonadotrophins with or without gonadotrophin-releasing hormone agonists/antagonists leads to a more effective ovarian response. Some of the goals of infertility treatment in the next century are to decrease the cost of medication and treatment and to reduce the number of multiple pregnancies. In this respect, the use of clomiphene citrate appears to be worth re-evaluating. The largest series of clomiphene patients, analysed by life-table or per- cycle methods, show that clomiphene is effective for at least six cycles of treatment. However, the combination of clomiphene with gonadotrophins or gonadotrophins only appear to be more effective than clomiphene alone.

The advent of recombinant gonadotrophins brought significant changes in fertility therapy. Treatment options with recombinant gonadotrophins add more to knowledge on folliculogenesis and ovarian steroidogenesis. Over a decade, recombinant LH (rLH) has been used for clinical trials, and the amount of peripheral LH that is necessary for optimal follicular growth, oocyte maturation, subsequent embryo development and assisted reproduction outcome during ovulation induction can now be better evaluated. This review evaluates the effect of rLH supplementation on ovarian stimulation and assisted reproduction outcome. The studies conducted with rLH supplementation in ovarian stimulation in different groups of patients and in cases of controlled ovarian stimulation are clearly discussed in this review.

The practice of ovulation induction and superovulation for IVF has progressed in many ways since the first reports of pregnancies after IVF over 25 years ago. The pharmacology of gonadotrophins has advanced alongside other developments, leading to safer, easier and more effective treatments for anovular infertility and IVF ovulation induction. This review uses current concepts of the basic physiology of gonadotrophin actions during the natural ovarian cycle as a starting point from which to assess their uses in anovulation and assisted reproduction. The relative merits of urinary and recombinant gonadotrophins are discussed, along with their uses in combination with gonadotrophin-releasing hormone agonists and antagonists.

For anovulatory women who fail to ovulate or conceive with clomiphene citrate, gonadotrophin ovulation induction has been the conventional second-line therapy. The aim of treatment is to achieve monofollicular development and ovulation. This differs fundamentally from the aim of ovarian stimulation for IVF, in which multiple follicular development is the goal. The small therapeutic window of ovulation induction requires a rigorous approach to monitoring, and willingness to cancel the cycle when multiple follicle development occurs. The two most widely used approaches are the low-dose step-up and the step-down protocols. While the latter more closely mimics the normo-ovulatory cycle, outcomes are similar. For safety reasons, the step-down protocol has not been widely adopted. The principle risks of ovulation induction are ovarian hyperstimulation syndrome and multiple pregnancy. There is a need to individualize treatment if outcomes are to be optimized. The role of adjuvant therapies remains unclear. However, prediction models based on initial screening parameters enable the optimal dose of FSH to be determined, and the identification of patients with a poor prognosis for successful treatment.

High hopes accompanied the release of gonadotrophin-releasing hormone (GnRH) antagonists onto the market at the end of the last millennium. Today, it must be admitted that not all of these hopes have been realized. According to large meta-analyses, treatment time for ovarian stimulation could be significantly shortened, and the incidence of severe ovarian hyperstimulation syndrome (OHSS) could also be reduced. However, the achieved clinical pregnancy rates seem not to be equivalent to those obtained after ovarian stimulation using GnRH agonists in the so-called long protocol. Very recent studies have demonstrated that oversuppression of LH after initiating GnRH antagonist administration seems not to be responsible for that observation. Moreover, supplementation with recombinant LH does not increase success rates. However, an analysis based on the data of the German IVF registry (DIR), scrutinizing more than 1800 cycles in so-called ideal patients (age < 35 years, first treatment cycle, pure tubal infertility, only classical IVF), did not demonstrate any differences in pregnancy rates between GnRH antagonists and GnRH agonists. These data seem to indicate that GnRH antagonists should be used as 'first choice treatment' in ovarian stimulation.

Different gonadotrophin preparations derived from human urine or manufactured by recombinant technology are currently used in clinical practice for the treatment of infertility. It has been widely assumed that gonadotrophin products manufactured by recombinant technology have better batch-to-batch consistency compared with human-derived preparations and that this potentially will be shown to provide a more constant clinical response, but there is little evidence for either statement. This study compared the batch-to-batch consistency between urinary-derived and recombinant manufactured gonadotrophin preparations using standard analytical techniques, as well as a novel in-vitro follicle bioassay to evaluate the consistency of the biological response at the target organ. Oligosaccharide isoform profiling, immunoassay testing, size exclusion chromatography analysis and in-vitro bioassay testing of urinary derived gonadotrophin preparations (MENOPUR and BRAVELLE) confirm that these products display a high degree of batch-to-batch consistency, similar to recombinant FSH (GONAL-f) either filled by mass or bioassay. The data also suggest that the batch-to-batch variation is independent of the manufacturing procedure (filled-by-bioassay or filled-by-mass) for the recombinant preparation (Gonal-f), but that the total FSH bioactivity delivered from a single dose preparation after reconstitution differs between the two manufacturing procedures.

A significantly lower pregnancy rate following the gonadotrophin-releasing hormone (GnRH) antagonist protocol as compared with the long GnRH agonist protocol has been reported. The objective of this study was to investigate whether increasing the dose of gonadotrophins on the day of antagonist administration would increase the pregnancy rate. This study is an open labelled, randomized controlled trial and allocation was done using sealed envelopes. One hundred and fifty-one subfertile couples undergoing IVF/intracytoplasmic sperm injection (ICSI) cycles were included in the study. Ovarian stimulation was started on day 3 of the cycle, using 150-300 IU human menopausal gonadotrophin (HMG)/day. From day 8 onward, daily vaginal ultrasound and daily urinary LH estimation were performed. If a premature LH rise was detected, the cycle was cancelled. The antagonist (0.25 mg daily) was started when the leading follicle reached 15 mm in mean diameter and LH testing in urine was negative up to and including the day of human chorionic gonadotrophin (HCG) injection. Patients were randomized on the day of starting the antagonist into two groups: group A, 72 patients with no increase in HMG dose, and group B, 79 patients in whom the dose of HMG was increased by 75 IU on the day of antagonist administration, and continued till the day of HCG administration. The results showed no statistically significant difference between the groups regarding number of oocytes retrieved, embryos obtained, implantation rate, clinical pregnancy rate and multiple pregnancy rate. It was concluded that there is no clinical evidence for increasing the dose of HMG on the day of antagonist administration.

Gonadotrophin treatment in clomiphene citrate resistant polycystic ovarian syndrome (PCOS) patients, using either low-dose step-up or low-dose step-down protocols, is highly effective to achieve singleton live births. Concomitant use of gonadotrophin releasing hormone analogues (GnRHa), which will block the endogenous feedback for monofollicular development during the low-dose step-up protocol, should not be employed. It is more difficult to induce ovulation in patients with more 'severe' PCOS, characterized by obesity and insulin resistance. There is need for optimization of starting doses for both the low-dose step-up and step-down protocols. Such optimization will prevent hyperstimulation due to a starting dose far above the FSH threshold, as well as minimize the time-consuming low-dose increments by starting with a higher dose in women with augmented FSH threshold. External validation of reported models for prediction of FSH response is warranted for tailoring and optimizing treatment for everyday clinical practice. Although preliminary, the partial cessation of follicular development, along with regression leading to atresia, lends support to the LH ceiling theory, emphasizing the delicate balance and need for both FSH and LH in normal follicular development. Future well-designed randomized controlled trials will reveal whether IVF with or without in-vitro maturation of the oocytes will improve safety and efficacy compared with classical ovulation induction strategies.

Immature oocyte retrieval followed by in-vitro maturation (IVM) is a promising potential treatment option, especially for women who are infertile through polycystic ovarian syndrome (PCOS). Although the pregnancy and implantation rates of IVM treatment are not as high as conventional IVF treatment, IVM treatment has many advantages for infertile women with PCOS, because this group of patients is extremely sensitive to stimulation with exogenous gonadotrophins and is at increased risk of developing ovarian hyperstimulation syndrome (OHSS). Different protocols have been used before immature oocyte retrieval, indicating that there are beneficial effects with FSH or LH priming on oocyte maturation. To date, the clinical pregnancy and implantation rates obtained from IVM treatment in infertile women with PCOS are approximately 30-35% and 10-15% respectively. Therefore, as an option, IVM treatment can be offered to women with PCOS instead of conventional IVF treatment with ovarian stimulation.

Salt-stored domestic cat oocytes are routinely used to study sperm function in domestic and nondomestic felids. Our objectives were to assess the effects of in vitro maturation (IVM), salt storage and storage duration on penetration of domestic cat oocytes by homologous spermatozoa. In Experiment 1, domestic cat spermatozoa were coincubated with fresh immature oocytes, salt-stored (2-3 weeks) immature oocytes, or salt-stored (2-3 weeks) IVM oocytes matured in Minimum Essential Medium containing 0.1IU FSH and 0.1IU LH/ml (IVM1) or 0.5IU FSH and 2.2IULH/ml (IVM2). In Experiment 2, all oocytes were matured (IVM2) and inseminated fresh or after salt storage for 2-3 weeks, 2-3 months or 9 months. In Experiment 1, penetration of the outer zona pellucida (OZP) was greater (P<0.05) in salt-stored IVM2 oocytes than in salt-stored immature oocytes, whereas penetration of salt-stored IVM1 oocytes was intermediate (P>0.05). In Experiment 2, penetration of the OZP and inner zona pellucida (IZP) was higher (P<0.05) in fresh IVM2 oocytes than in salt-stored oocytes, and a higher (P<0.05) proportion of oocytes had IZP sperm after 2-3 weeks of storage than after 2-3 months. Penetration of the perivitelline space was higher (P<0.05) in fresh IVM2 oocytes than in oocytes stored for 2-3 weeks or 2-3 months. These results suggest that oocyte penetration is improved by IVM, but is impaired by exposure to salt-storage solution and prolonged storage duration.

Modern gonadotrophin-releasing hormone (GnRH) antagonists such as cetrorelix and ganirelix reliably prevent premature LH surges in controlled ovarian hyperstimulation for assisted reproductive technologies. Cetrorelix and ganirelix are safe and effective compounds. Because of their distinct pharmacological mode of action, it has been possible to achieve a significant reduction of treatment time. Fertilization and pregnancy rates are comparable to those obtained in agonist protocols for ovarian stimulation. No allergic or hyperergic reactions have been reported, and patient compliance is excellent. The fact that GnRH antagonists allow an immediate suppression of gonadotrophin concentrations while preserving pituitary responsiveness to endogenous GnRH provides enormous flexibility in treatment. GnRH antagonists have helped to overcome some major disadvantages of GnRH agonists, especially of the long protocol, which is currently the standard protocol for ovarian stimulation. The mistaken administration of antagonists during early pregnancy is not possible, since they are only administered during ovarian stimulation when a premature LH surge may be imminent. They are used in the spontaneous cycle or after pretreatment with oral contraceptives. Pregnancy can easily be ruled out by testing for human gonadotrophic hormone before onset of gonadotrophin stimulation on the second or third day of the cycle. Since flare-up effects are absent, there is no risk of cyst formation. Hormonal withdrawal symptoms are eliminated, since no period of pituitary suppression occurs, and therefore exogenous gonadotrophins are not required. Overall, the duration of the stimulation cycle is as short as a normal menstrual cycle. The procedure seems to be safer than the long protocol, since the most serious complication, the occurrence of severe cases of OHSS, is reduced. It is also safe with respect to the course of pregnancies and the health of offspring. Both protocols developed so far, the single dose and multiple dose antagonist protocol, are comparable, utilizing data from the large prospective phase IIIb studies. Although several studies have indicated a slight reduction in pregnancy rate with GnRH antagonists as compared with agonists, this problem may be rectified by developing flexible antagonist regimens designed for individual patients. Introducing flexible GnRH antagonist regimens should be the area of research in the near future.