Ovarian Stimulation Treatment Research Articles

Value of early referral to improve fertility preservation outcomes in women with breast cancer.

9026 Background: ASCO recommends that young cancer patients be referred to specialists for fertility preservation (FP) as soon as possible after the diagnosis, however, there is no data to directly support that recommendation. The purpose of this study was to determine if early referral improves FP outcomes in young women with breast cancer undergoing adjuvant chemotherapy and/or radiotherapy. Methods: We performed a secondary analysis of a prospective database (Controlled Ovarian Stimulation Treatment with Letrozole Supplementation Study, COST-LESS, JCO 2009) of 154 women with breast cancer undergoing ovarian stimulation (OS) for FP by embryo or oocyte cryopreservation. Complete data were available in 93 patients with mean age of 35.2±4.4 years (range: 24 to 44). Results: Thirty-six of the 93 patients were referred and evaluated prior to breast surgery (PreS) and 57 post-surgery (PostS). PreS and PostS groups were similar in terms of age, cancer stage, and receptor or BRCA status. Likewise the mean number of oocytes or embryos cryopreserved per cycle were similar between the two groups. The time period from the first diagnosis date (FD) to initiation of OS was significantly shorter in patients who were referred PreS vs. PostS (43.0±28.0 vs. 72.5±30.6 days, p<0.001, Mann Whitney U test). There was, however, no difference in the length of time from FD to surgery date and from the date of first FP consultation to initiation of OS between the two groups. Six of 36 patients in the PreS group and 1 of 57 in the PostS group were able to undergo two FP cycles (p = 0.012, Fisher's exact test). This resulted in an additional 79 vs. 5 oocytes obtained in PreS vs PostS groups respectively. Patients who underwent first oocyte retrieval within 5 weeks of the surgery were able to complete a second cycle within 9 weeks of the surgery. Conclusions: Early referral prior to breast cancer surgery enables multiple cycles of FP without a delay in chemotherapy beyond 9 weeks post surgery. Women who can undergo multiple cycles may be at an advantage for FP because of additional number of eggs/embryos cryopreserved. This is the first study demonstrating the benefit of early FP referral in cancer patients. Practitioners should adhere to FP guidelines issued by ASCO in 2006. No significant financial relationships to disclose.

328 IN VITRO MATURATION OF OOCYTES FROM Canindé GOATS SUBMITTED TO DIFFERENT HORMONAL TREATMENTS FOR OVARIAN STIMULATION

Oocyte IVM is a long process during which oocytes acquire their ability to support the stages of development in a stepwise manner, ultimately reaching activation of the embryonic genome. The overall success of this process can be affected by factors such as hormonal treatment for ovarian stimulation. Thus, the current study aims to evaluate the possible effects of the ovarian stimulatory protocols on the goat oocyte quality and IVM rate. Adult and cyclic Canindé goats were heat-synchronized by means of intravaginal sponges impregnated with 60 mg medroxyprogesterone acetate (MAP, Progespon, Syntex, Buenos Aires, Argentina) inserted for 11 days coupled with a luteolytic injection of 50 μg cloprostenol (Ciosin, Coopers, São Paulo, Brazil) in the 8th day of treatment. The ovarian stimulation was carried out using one of the following protocols: a) standard multi-doses (MD) with 120 mg pFSH (Folltropin-V, Vetrepharm, Canada) distributed in five injections (30/30; 20/20; 20 mg) at 12 h intervals (n = 18); b) three- doses (TD) with 120 mg pFSH administered in three injections (60; 40; 20 mg) at 24 h intervals (n = 17); c) one shot (OD) of 70 mg pFSH plus 200 IU of eCG (Novormon, Syntex) administered 36 h before sponge removal (n = 17). In MD andTD groups, the pFSH injections started in Day 8 of progestagen treatment. The follicles were aspirated just after the sponge removal using laparoscopic oocyte recovery (LOR). This procedure was performed with a 22-gauge needle and a vacuum pump at 30 mmHg. The collection medium was TCM-199 supplemented with HEPES (10 mM), heparin (20 IU mL-1), and gentamicin sulfate (40 μg mL-1). COCs were classified as grade I, II, III, or IV based on visual criteria (Baldassarre H et al. 2003 Theriogenology 56, 831-839). Good quality oocytes (grade I and II) were incubated in TCM-199 supplemented with cysteamine (100 μM), EGF (10 ng mL-1) and gentamicin sulfate (40 μgm L-1) at 38.5°C in a humidified atmosphere with 5% CO2 in air for 24 h. Oocyte maturation was assessed by the visualization of first polar body under inverted microscope. Data were expressed as percentages and analyzed using the Fischer’s exact test. No statistical differences among hormonal treatments (P &gt; 0.05) were observed for the percentage of the good quality oocytes, with 70.4 ± 3.0% of COCs graded in I and II. The IVM rate inTD (31.4%) was statistically lower than MD (31.4% v. 46.5%, P = 0.04) group. However, no significant differences (P = 0.89) were observed between OD (45.2%) and MD groups. Thus, current results indicate that oocyte production for IVM can be facilitated using ovarian stimulation with the one shot FSH/eCG regime without affecting meiotic competence. In summary, OD and MD treatments can be used for oocyte IVM in an embryo production programme in Canindé goats. This study was supported by the following Brazilian agencies: FINEP, CNPq, FUNCAP, and CAPES.

Effect of repeated gonadotropin stimulation on ovarian reserves and proliferation of ovarian surface epithelium in mice

This study aimed to evaluate the effect of repeated ovarian stimulation (OS) on the ovarian follicular population and morphology in female mice and its influence on the embryo’s developmental ability, and the profile of the ovarian surface epithelium (OSE). A total of 75 mice were enrolled in this experiment and randomly assigned into three groups: repeated ovarian stimulated group [n = 25; receiving 5 IU pregnant mare serum gonadotrophin (PMSG) and human chorionic gonadotropin (hCG) at 6 day intervals for 5 cycles]; single ovarian stimulated group (n = 25; receiving 5 IU PMSG and hCG for 1 cycle), and control group (n = 25; without additional treatment). The follicle number at various stages and the morphologies were recorded respectively in the three groups. The harvested oocytes or embryos, cleavage rate, good quality embryo rate, and blastocyst production rate were counted and calculated, and the proliferations of ovarian surface epithelium were evaluated respectively. In the three groups, the single ovarian stimulation treatment significantly increased the mean number of ovarian oocytes or embryos (39.25±10.77 one-cell embryos/female); on the other hand, repeated gonadotropin stimulation obtained the lowest mean number (5.15± 2.81 eggs/female, P<0.01). Repeated ovarian stimulation also tended to decrease normal follicles of primary follicles (66.67%) and secondary follicles (72.86%), and got the lowest cleavage rate (67.47%), lowest good quality embryo rate (2.41%), and lowest blastocyst production rate (0). The OSE cells adjacent to the antral follicles and corpus luteum (CL) in the repeated ovarian stimulated group (81.8%) had a significantly higher proliferation rate than the other groups. The proliferation rate of the OSE in the single ovarian stimulated group (56.4%) was significantly higher than that in the control group (37.5%) (P<0.01). In conclusion, single ovarian stimulation may produce more oocytes/embryos. However, repeated gonadotropin stimulation may have a negative effect on the ovarian follicular quality, the number of mature retrieved oocytes, and the embryo quality, even increasing the chance of ovarian cancer.

Intrauterine insemination

Intrauterine insemination (IUI) with or without ovarian stimulation is a common treatment for infertility. Despite its popularity, the effectiveness of IUI treatment is not consistent, and the role of IUI and in vitro fertilization (IVF) treatment in practice protocols has not been clarified. Medline searches were done by individual topics (utilization, procedures, effectiveness of partner but not donor IUI and related endocrine issues). Effectiveness of IUI was evaluated in relevant randomized controlled trials, using meta-analysis and meta-regression where necessary. Stimulated IUI is ineffective in male infertility and the effect on other diagnoses is small. With clomiphene citrate and IUI, the most common IUI protocol, pregnancy rates average 7% per cycle. FSH ovarian stimulation and IUI treatment is only modestly better than observation only with pregnancy rate 12% per cycle but multiple birth rates averaging 13%. Mildly stimulated (1-2 follicles) cycles might reduce the cost and multiple birth rates but may require more cycles of treatment. Prevention of premature luteinizing hormone surges and luteal phase support do not appear to be major requirements in IUI cycles. IUI treatment requires ovarian stimulation to achieve modest results, but the high multiple pregnancy rates mean that it is no more than a poor substitute for IVF treatment. More trials are needed on IUI treatment with mild stimulation and on the order of IUI and other treatments.

Understanding the burden of ovarian stimulation: fertility expert and patient perceptions

This multinational, interview-based study was conducted to provide insights into participants' experiences, behaviour, attitudes and emotions towards fertility treatments. One study group comprised 185 patients who were undergoing ovarian stimulation for IVF while the other comprised 170 physician and nurse fertility experts. A key study objective was to identify which aspects of ovarian-stimulation treatment contribute to the physical and psychological burden on patients. Ovarian-stimulation treatment was considered by 55% ( n = 102) of patients to impact upon daily life, while 31% ( n = 57) felt that daily injections limited their everyday activities. The most frequent questions from patients to physicians and nurses concerned fertility drug-related side effects, followed by concerns about how to perform their injections. More than half of patients (57%, n = 106) had concerns about the injection process. Within the fertility expert group, almost half (47%, n = 80) expressed concern about whether their patients injected themselves correctly, with 26% ( n = 44) raising concerns about patient compliance. This study adds to the growing body of literature on the impact of ovarian stimulation on patients' lives and highlights areas in which improved patient education could help to reduce the psychological burden of IVF treatment.

Benefits of luteinizing hormone activity in ovarian stimulation for IVF

Knowledge of the physiology of folliculogenesis has led to a better understanding of the role of LH activity in this process. This role has been investigated in the setting of ovarian stimulation and several treatment modalities have been proposed. LH activity can be provided in the forms of: (i) human menopausal gonadotrophins (HMG), (ii) recombinant LH (rLH), (iii) human chorionic gonadotrophin (HCG), and (iv) recombinant HCG (rHCG). Current evidence suggests that ovarian stimulation performed with HMG in patients treated with a long gonadotrophin releasing hormone (GnRH) agonist protocol seems to result in increased (3–4%) clinical pregnancy rates compared with recombinant FSH (rFSH). Data regarding the short GnRH agonist or GnRH antagonist protocol are scarce and firm conclusions cannot be drawn. Administration of rLH prior or during ovarian stimulation with rFSH does not seem to increase the probability of pregnancy. Existing data suggest that HCG is capable of partially or completely replacing FSH administration during the mid-to-late follicular phase of an ovarian stimulation cycle, leading to a decreased requirement for FSH, without compromising pregnancy rates. High quality evidence originating from randomized controlled trials regarding the potential value of rHCG administration during ovarian stimulation for IVF is not available.

Incidence of embryo aneuploidies in natural and stimulated cycles in the same oocyte donor. Preliminary results

OBJECTIVE: To compare embryo aneuploidies rate between natural and stimulated cycles. DESIGN: Prospective intra-patient comparative study. MATERIALS AND METHODS: Patients: Oocyte donors aged 18 to 35. -Inclusion criteria: Regular menstrual cycles; no previous ovarian stimulation treatments. -Exclusion criteria: endometriosis, policystic ovarian syndrome; recurrent miscarriages. All donations were performed under the following conditions: -Recipients aged until 45 years old following egg and sperm donation. -Recipients with background of uterine disease or recurrent miscarriages were excluded. Outcome measures: Primary: Embryo aneuploidies in natural vs. stimulated cycles in the same egg donor; Secondary: Pregnancy, implantation and miscarriage rates. Protocol: Firstly, the donor underwent a non stimulated cycle. When the follicle reached 18 mm in diameter, rCG was administered and oocyte retrieval was scheduled 36 h later. The resulting embryo underwent preimplantation genetic diagnosis using fluorescence in-situ hybridization analysis with probes for chromosomes X,Y, 13,15,16, 18, 21 and 22. If the embryo could not be analyzed, the donor was dropped out of the study. Euploid embryos were transferred on day 5. Secondly, donors followed ovarian stimulation under a GnRH agonist long protocol and a starting fixed dose of 150 UI of rFSH and 75 UI of hp-hMG. Oocyte retrieval was programmed when three or more follicles reached 18 mm in diameter. Recovered oocytes were inseminated with the same sperm donor. All the embryos from the stimulated cycle were also analyzed and a maximum of 2 euploid embryos were transferred. RESULTS: Are shown in table 1.Table 1Natural cycle (n=21)Stimulated cycle (n=18)pOR (95% CI)Aneuploid rate52.4 (29.8-74.3)49.1 (39.2-58.9)0.780.87 (0.34-2.23)Pregnancy rate30.0 (6.7-65.2)80.0 (51.9-95.7)0.019.33 (1.46-59.48)Implantation rate20.0 (2.5-55.6)40.0 (22.7-59.4)0.262.67 (0.48-14.79)Ongoing pregnancy rate10.0 (2.5-44.5)46.7 (21.3-73.4)0.067.87 (0.79-78.67)Pregnancy loss rate66.7 (9.4-99.2)33.3 (9.9-65.1)0.314.00 (0.27-58.56) Open table in a new tab CONCLUSIONS: A similar aneuploidies rate is observed in the natural versus stimulated cycle in young normoovulatory women. Ovarian stimulation does not increase the incidence of chromosomally abnormal embryos in this group of patients. Pregnancy rate was significantly higher in stimulated cycles.

Controlled Ovarian Stimulation Induces a Functional Genomic Delay of the Endometrium with Potential Clinical Implications

Controlled ovarian stimulation induces morphological, biochemical, and functional genomic modifications of the human endometrium during the window of implantation. Our objective was to compare the gene expression profile of the human endometrium in natural vs. controlled ovarian stimulation cycles throughout the early-mid secretory transition using microarray technology. Microarray data from 49 endometrial biopsies obtained from LH+1 to LH+9 (n=25) in natural cycles and from human chorionic gonadotropin (hCG) +1 to hCG+9 in controlled ovarian stimulation cycles (n=24) were analyzed using different methods, such as clustering, profiling of biological processes, and selection of differentially expressed genes, as implemented in Gene Expression Pattern Analysis Suite and Babelomics programs. Endometria from natural cycles followed different genomic patterns compared with controlled ovarian stimulation cycles in the transition from the pre-receptive (days LH/hCG+1 until LH/hCG+5) to the receptive phase (day LH+7/hCG+7). Specifically, we have demonstrated the existence of a 2-d delay in the activation/repression of two clusters composed by 218 and 133 genes, respectively, on day hCG+7 vs. LH+7. Many of these delayed genes belong to the class window of implantation genes affecting basic biological processes in the receptive endometrium. These results demonstrate that gene expression profiling of the endometrium is different between natural and controlled ovarian stimulation cycles in the receptive phase. Identification of these differentially regulated genes can be used to understand the different developmental profiles of receptive endometrium during controlled ovarian stimulation and to search for the best controlled ovarian stimulation treatment in terms of minimal endometrial impact.

Obstetric outcomes following vitrification of in vitro and in vivo matured oocytes

To evaluate obstetric outcomes with oocyte vitrification after ovarian stimulation (OS) and in vitro maturation (IVM) of immature oocytes. A prospective trial from October 2003 to April 2007. University-based medical center. OS group: 38 patients undergoing intrauterine insemination who overresponded to OS. IVM group: 20 patients who had previous unsuccessful intrauterine insemination. Mature oocyte retrieval following OS. Immature oocyte retrieval and IVM. Oocyte vitrification, thawing, insemination, and transfer of the resulting embryos. Live-birth rates and obstetric outcomes. The OS group was superior to the IVM group in terms of oocyte survival (81.4 +/- 22.6% vs. 67.5 +/- 26.1%), fertilization rate (75.6 +/- 22.5% vs. 64.2 +/- 19.9%), and cumulative embryo score (38.4 +/- 22.3 vs. 20.0 +/- 13.8). However, the differences in the implantation rate per embryo (19.1 +/- 25.8% vs. 9.6 +/- 24.1%), clinical pregnancy rate per cycle started (44.7%, vs. 20.0%), and live-birth rate per cycle started (39.5% vs. 20.0%) were not statistically significant. Twenty healthy babies were born in the OS group and four in the IVM group. Pregnancies achieved with vitrification of oocytes after OS and IVM treatments do not appear to be associated with adverse pregnancy outcomes. Vitrification of IVM oocytes represents a novel option for fertility preservation.

Doppler assessment of uterine artery blood flow for the prediction of pregnancy after assisted reproduction treatment

Doppler assessment of uterine artery blood flow for the prediction of pregnancy after assisted reproduction treatment

Ultrasonographic-guided retrieval and in vitro maturation of eland ( Taurotragus oryx) and bongo ( Tragelaphus eurycerus isaaci) antelope oocytes

The limited availability of gametes is a major factor hindering the development and application of assisted reproductive technologies (ART) in large non-domestic ungulates. This is partly due to the small number of captive animals and handling difficulties associated with procedures for gamete recovery. In the present study, results are reported of multi-year studies on ovarian stimulation and oocyte retrieval by ultrasonographic-guided transvaginal follicular aspiration and subsequent in vitro maturation (IVM) in eland and bongo antelopes. All procedures were conducted on sedated females handled in a hydraulic chute without inducing general anesthesia. Five estrous synchronization/ovarian stimulation protocols were evaluated and data are presented on 73 and 15 procedures in eland and bongo, respectively. Repeating procedures (≤once/month) on the same female did not affect ovarian response or number oocytes recovered in either species. Eland females, but not the ovarian stimulation treatment, affected ovarian response. Ovarian stimulation treatment affected oocyte recovery rate in eland, but not in bongo. In both species, ovarian hormone stimulation treatment affected the distribution of follicles by size and the status of expansion of the cumulus cell investment of oocytes, but not the frequency of metaphase II oocytes during IVM. The timing of extrusion of the first polar body during IVM was more synchronous in bongo than in eland oocytes. It is concluded that Transvaginal oocyte retrieval (TVOR) can be safely and repeatedly applied in gonadotropin-treated eland and bongo females to recover oocytes that can mature in vitro. The methods described for the present study can be adapted to improve the availability of non-domestic ungulate oocytes for basic and applied studies.

Molecular pathology of the FSH receptor: New insights into FSH physiology

Manipulations of mouse genome have helped to elucidate gonadotrophin function but important differences subsist between rodent and human reproduction. Studies of patients with mutations of gonadotrophins or gonadotrophin receptors genes allow understanding their physiological effects in humans. The correlation of the clinical phenotypes of patients with in vitro studies of the mutated receptor residual function and histological and immunohistological studies of the ovarian biopsies permits to understand which stages of follicular development are under FSH control. Total FSH receptor (FSHR) inactivation causes infertility with an early block of follicular maturation remarkably associated with abundant small follicles as in prepubertal ovaries and demonstrates the absolute requirement of FSH for follicular development starting from the primary stage. Partial FSHR inactivation, characterized by normal-sized ovaries, can sustain follicular development up to the early antral stages but incremental levels of FSH stimulation seem to be required for antral follicular growth before selection. These findings contrast with the traditional view of an initial gonadotrophin-independent follicular growth prior to the preantral–early antral stages. The presence of numerous reserve follicles in the ovaries of these patients may permit a future treatment of their infertility. The study of reduced FSHβ or FSHR activity in genetically modified male mice models and in men suggests a minor impact of the FSHR on masculine fertility. Further studies on patients with a demonstrated total FSHβ or FSHR inactivation are required to elucidate reported differences in spermatogenesis impairment. Finally, the studies of mutations of gonadotrophins and their receptors demonstrate differences in gonadotrophin function between genetically modified rodents and humans which suggest prudence in extrapolating observations in rodents to human reproduction. Ovarian hyperstimulation syndrome (OHSS) can infrequently arise spontaneously during pregnancy, but most often it is an iatrogenic complication of ovarian stimulation treatments with ovulation drugs for in vitro fertilization. The first genetic cause of familial recurrent spontaneous OHSS was identified as a broadening specificity of the FSHR for hCG due to naturally occurring heterozygous mutations located unexpectedly in the transmembrane domain of the FSHR. Broadening specificity of a G protein-coupled receptor is extremely rare. These observations led to the identification of the etiology of this previously unexplained syndrome and permitted to conceive novel models of FSHR activation. Susceptibility to iatrogenic OHSS or its clinical severity may be associated with FSHR polymorphisms with slightly different activities in vivo as suggested by several studies. The study of larger cohorts is needed to evaluate the clinical impact of these observations in the management of patients undergoing IVF protocols.

Does assisted reproduction therapies with gonadotrophins induce body weight variations? a prospective study

During the controlled ovarian stimulation (COS) treatment, nursing staff detect a high number a patients with apprehensiveness for the possibility of weight augmentation. In this study we try to analyse if there is a body weight fluctuation and if it's related to any paramether other than the gonadotrophins administration.

Features of follicle-stimulating hormone–stimulated follicles in a sheep model: keys to elucidate embryo failure in assisted reproductive technique cycles

To evaluate the individual functionality of gonadotropin-stimulated preovulatory follicles, for understanding embryo failure in assisted reproductive technique cycles, in a sheep model. Observational, model study. Public research unit. Fifteen adult Manchega ewes. Synchronization of the estrous cycle with intravaginal progestagens and ovarian stimulation with FSH; evaluation of reproductive activity, plasma sampling, ovarian ultrasonography, and ovariectomies. Determination of estrus behavior, plasma and intrafollicular concentrations of E(2) and inhibin A, number and size of ovarian follicles, and developmental competence of oocytes. These results support the usefulness of serial measurements of plasma inhibin A for assessment of follicular growth during the FSH treatment, rather than of E(2) assays commonly used. Functionality of FSH-stimulated preovulatory follicles is clearly disturbed, as confirmed by a negative correlation between follicular size and intrafollicular concentrations of inhibin A and E(2) in preovulatory follicles after individual dissection; moreover, the ability of their oocytes to resume meiosis was diminished. Functionality of follicles in controlled ovarian stimulation (COS), and developmental competence of their oocytes, is disturbed by the high doses of gonadotropin supplied and finally determined by follicular sizes at starting FSH treatment.

Global epidemiology of multiple birth

Multiple births, which account for approximately 3% of births and 14% of infant deaths, are increasing in frequency. Multiple birth rates began to decline in the 1950s, reaching a minimum in the 1970s and rising since then. Both twin and triplet rates followed the same rising trend until 1998, after which triplet birth rates began to decline while twin birth rates continued to rise. Rising maternal age is associated with rising frequency of dizygotic twinning up to 37 years of age. Older maternal age, associated with the social trend to delayed child bearing, accounts for 25–30% of the rise in multiple birth rates since 1970. The resulting rise in the prevalence of infertility has given rise to unprecedented use of ovarian stimulation treatments that stimulate the development of multiple oocytes. Assisted reproduction technology and ovulation stimulation with clomiphene citrate or gonadotrophins without assisted reproduction account for similar proportions of both twin births (20–30%) and triplet births (30–40%). The fall in triplet rates since 2000 is reassuring, but fetal reduction of high order pregnancies may be a factor in rising twin rates. Continuing attention is needed to all possible means of minimizing triplet pregnancies.

Evaluation of functional gene polymorphisms in the FSH receptor of patients with Ovarian Hyperstimulation Syndrome (OHSS)

OHSS most often occurs as a complication of ovarian stimulation treatments for in vitro fertilization (IVF) and is accompanied with increased polycystic ovaries, edemas and ascites. The incidence of OHSS ranges from 0.6–14%, with severe OHSS cases ranging from 0.2–5%. The clinical manifestations vary from abdominal distension to potentially life-threatening, massive ovarian enlargement and capillary leak with fluid sequestration.

O-172: Human cumulus cells gene expression profile according to ovarian hyperstimulation protocols (hMG vs. recFSH)

The emergence of DNA chips offers the possibility to exhaustively catalogue the genes that are expressed in a given tissue at a given time. The individual characterization of gene activity at a genomic scale in cumulus cells of each patient will provide important insights to evaluate the effects of ovarian stimulation treatments under IVF conditions. We have studied the cumulus cells RNA expression profile in order to identify the molecular markers differentially regulated following recFSH and hMG stimulation protocols. Total RNA of cumulus cells collected from patients undergoing an intracytoplasmic sperm injection (ICSI) in our centre and stimulated with hMG (group G1) or recFSH (group G2) were extracted. The RNA expression profiles were evaluated on DNA microarray chips. Total genes expressed and genes differentially expressed between these two groups were quantified and analyzed. The patients referred for ICSI were stimulated with a combination of gonadotropin-releasing hormone agonist (GnRH-a) and recombinants recFSH or hMG. Cumulus cells were mechanically removed on the day of egg collection and frozen at -80°C in RLT buffer from the RNeasy kit (Qiagen) before RNA extraction. Pools of cumulus cells from a single patient were analyzed on Affymetrix™ HG-U133 plus 2.0 GeneChip oligonucleotide array chips made of 54675 probesets and representing roughly 35000 unique human known or predicted genes. The Affymetrix™ GeneChip Operating Software 1.2 (GCOS) was used to evaluate the signal intensity for each probeset (detection call ”present“ or ”absent“) and also to perform pair wise comparison between single gene expression raw values from cumulus cells of different stimulation protocoles. We determined genes exclusively or differentially expressed between the two groups. Differential expression was determined by selecting the probesets with the following constraints: ratios ≥ 3 with a significant change p-value (P ≤ 0.01), ”overexpressed“ probeset ”present“ with a signal value ≥ 20. A total of 11932 genes are present in both groups. 2341 and 1476 are exclusive to G1 and G2 respectively. However, only 64 and 260 genes represent a significant difference between the two groups. Most gene expression levels (n= 31811) do not change between the two groups. Among the genes that are expressed in the two groups, 518 are upregulated in G1 whereas 1742 are upregulated in G2. In G1, the LHCGR gene represents one of the strongest difference with a 6.7 fold increase in cumulus cells under hMG when compared with recFSH. In addition, other genes of interest include CD163 and PAPPA, upregulated with hMG, and GREM1 and CDW92 with recFSH. We identified human genes that are differentially expressed in patients that were undergoing distinct stimulation protocols. Understanding the relationships between ovarian stimulation and cumulus cell gene expression profile will permit to adapt stimulation protocols in order to elicit an adequate response benefiting the IVF outcome.

Cumulative pregnancy rates resulting from the use of fresh and frozen oocytes: 7 years’ experience

Storing supernumerary embryos and transferring them later fully utilizes the reproductive potential of retrieved oocytes, allowing a significant increase in the overall number of pregnancies achieved from a single cycle of ovarian stimulation treatment. As an alternative to embryo cryopreservation, preservation of unfertilized oocytes has been proposed to maximize clinical outcome. This paper presents data concerning the cumulative pregnancy rate after use of fresh and cryopreserved oocytes. In 80 treatment cycles in which patients chose to have only a few fresh oocytes inseminated, 24 pregnancies were obtained (30.0%), with an implantation rate of 22.6%. After cryopreservation with the standard slow-cooling protocol, the survival, fertilization and cleavage rates of 918 frozen oocytes were 43.4, 51.5 and 86.0% respectively. A total of 14 frozen pregnancies were achieved, with pregnancy rate 19.2% per transfer and implantation rate 12.3%. The cumulative pregnancy rate was 47.5% per patient. Therefore, despite a low rate of oocyte post-thaw survival, it appears that oocyte storage appreciably improves the number of pregnancies per treatment cycle in cases in which only a minority of oocytes are destined for the fresh treatment. This outcome provides valuable information for appraising the chances of clinical success when the option of embryo cryopreservation is not available.

274 LAPAROSCOPIC OVUM PICK-UP IN SHEEP AND GOATS: EFFECTS OF REPEATED RECOVERIES AND FOLLICULAR DIAMETER

Laparoscopic ovum pickup (LOPU) is a modern technique that may be used in programs of sheep and goat in vitro embryo production. The effects of repeated recoveries and follicular diameter on the efficiency of LOPU were evaluated in these species. In six Merino donor sheep and five Criolla goats, ovarian stimulation for follicular development was performed by administering a single dose of 60 mg NIH-FSH-P1 (Folltropin®; Bioniche, Canada) and 300 UI of eCG (Novoromon 5000®; Syntex, Argentina). Every four days, a total of 4 LOPU in sheep and 3 in goats were performed 24 h after each ovarian stimulation treatment. The intravaginal sponge with 60 mg of medroxyprogesterone (Esponjas Vaginales Syntex®; Syntex, Argentina) were removed after the last LOPU. Follicular fluid was aspirated under laparoscopic observation according to the follicle diameter with a cannula for intrauterine artificial insemination without a vacuum pump. The number and size of follicles, oocyte quantity and quality and recovery rate were evaluated. A total of 566 follicles were aspirated (14.4 ± 2.4 follicles/sheep and 14.6 ± 7.5 follicles/goat), yielding a total of 263 oocytes (6.8 ± 1.5 oocytes/sheep and 6.6 ± 2.9 oocytes/goat) and a recovery rate of 47% in sheep and 46% in goats (Table 1). In both species, there were no significant differences in the number of aspirated follicles, recovered oocytes, overall recovery rate, and the recovery rate of good quality oocytes in the repeated LOPU. The oocyte recovery rates in sheep differed significantly according to the follicular size (55% in large follicles, 36% in small follicles; P &lt; 0.05), whereas in goats recovery rates of oocytes related to follicular size showed no significant differences (42 and 54% for large and small follicles, respectively). Follicular sizes from which oocytes were obtained was not a determining factor in the recovery rate of good quality oocytes. The technique used would allow effective and repeated oocyte recoveries for in vitro embryo production in sheep and goats. Table 1. Repeated recoveries by laparoscopic ovum pickup (LOPU) in sheep and goats

Presence and Absence of Follicle-Stimulating Hormone Receptor Mutations Provide Some Insights into Spontaneous Ovarian Hyperstimulation Syndrome Physiopathology

Ovarian hyperstimulation syndrome (OHSS) is a potentially life-threatening complication of ovarian stimulation treatments. Moreover, four mutations of the FSH receptor (FSHr) were recently described in patients presenting with spontaneous OHSS (sOHSS) of the first trimester of pregnancy with normal levels of human chorionic gonadotropin (hCG). The objective of this study was to look for novel FSHr mutations in patients with sOHSS associated with different levels of hCG and TSH to 1) find new residues important for FSHr activation and specificity, and 2) better delineate the pathophysiology of the different presentations of sOHSS. DESIGN, INTERVENTION, AND PATIENTS: After blood sampling, we sequenced the FSHr from genomic leukocytes DNA from eight patients with sOHSS of the first or second trimester of pregnancy with normal or high hCG levels or with high TSH levels associated with severe hypothyroidism. This study was performed at a university laboratory. The main outcome measure was FSHr sequencing and in vitro evaluation of the variation of cAMP production of FSHr mutants. A new mutation was found in the patient with sOHSS of the first trimester of pregnancy with a normal hCG level: I5.54(545)T, in transmembrane helix V of the FSHr. When tested functionally, this mutant displayed promiscuous activation by both hCG and TSH together with detectable constitutive activity. In contrast, no mutations were found in the FSHr from patients with high hCG or TSH levels, indicating that for those seven patients, sOHSS results from the natural promiscuous stimulation of a wild-type FSHr by very high concentrations of hCG or TSH. sOHSS can occur by at least three different pathophysiological mechanisms.