Development Of Multiple Follicles Research Articles

Childhood growth of singletons conceived following intracytoplasmic sperm injection – irrelevance of gonadotropin stimulation

BackgroundIn conventional, gonadotropin stimulated, in vitro fertilization or intracytoplasmic sperm injection (c-IVF/ICSI) growth and development of multiple follicles is induced by gonadotropins, combined with gonadotropin-releasing hormone agonist or antagonist. In recent studies, singletons conceived after c-IVF/ICSI cycles had lower birth weight not only than spontaneously conceived children but also children born after unstimulated natural IVF/ICSI cycles (NC-IVF/ICSI). Lower birth weight is associated with a catch-up growth within the first years of life. Following the Barker hypothesis accelerated growth has been associated with a higher risk of cardiovascular diseases later in life. The aim of the study is to assess, if children conceived with NC-IVF/ICSI have a higher birthweight and therefore do not show a catch-up growth within the first two years. Therefore, we assume that children born after NC-IVF/ICSI have a better long-term cardiometabolic risk profile. Whether the weight- and height gain is comparable to spontaneously conceived children is unknown, since to our knowledge we are the first study to investigate the longitudinal growth of children born after unstimulated natural cycle ICSI (NC-ICSI).Material and methodsWe conducted a single-center, prospective cohort study (2010-2017) including children (n = 139) born after NC-ICSI or c-ICSI treatment. Growth parameters up to 24 months were collected. Standard deviation scores based on growth references were calculated.ResultsThe study included 98 children in the NC-ICSI and 41 children in the c-ICSI group. The median birth weight in NC-ICSI children was 3.4 kg [0.1 standard deviation score (SDS)] compared to 3.3 kg (−0.3 SDS) in c-ICSI children (p = 0.61). Median length at birth was 50 cm in both groups (NC-ICSI (−0.5 SDS), c-ICSI children (−0.8 SDS), p = 0.48). At age 24 months, median weight in NC-ICSI children was 12.2 kg (0.3 SDS) versus 12.2 kg (0.2 SDS) in c-ICSI children (p = 0.82) and median length 87.5 cm (0.1 SDS) versus 88.0 cm (0.4 SDS) (p = 0.43).ConclusionWe found no difference in growth between children conceived after stimulated and unstimulated ICSI. Growth parameters of both treatment groups did not differ from Swiss national growth references (N = 8500). One of the main limitations of our study was the small sample size (N = 139) of complete data sets over time and the high drop-out rate of 49% (68/139). Nevertheless, with the increasing number of children born after IVF/ICSI every year it is of immense importance to search for possibilities to reduce their long-term cardiometabolic risk and we want our data to contribute to this discussion.

Correlation between the Total Dose of Follicle-Stimulating Hormone and Cumulative Live Birth Rate in Women Undergoing in Vitro Fertilization

Background: Follicle-stimulating hormone (FSH) is widely used during assisted reproductive therapy to facilitate the development of multiple follicles. In turn, the number of oocytes retrieved has been shown to relate to the live birth rate. This retrospective study investigated the correlation between the total FSH dose used and the cumulative live birth rate in individuals who had undergone in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI). Methods: This retrospective study included women undergoing IVF/ICSI using a gonadotropin-releasing hormone antagonist protocol who had a regular menstrual cycle and an expected normal response to ovarian stimulation. Cumulative live birth rate was defined as the first live birth following the use of all fresh and frozen transfers of embryos derived from one stimulated IVF/ICSI cycle. Results: The analysis included 7,130 stimulated cycles in 6,517 individuals (9,138 transfers). The mean age, antral follicle count, and anti-Müllerian hormone level were [Formula: see text], [Formula: see text], and [Formula: see text][Formula: see text]ng/ml, respectively. The mean total FSH dose, total number of oocytes retrieved, and total FSH dose per oocyte retrieved were [Formula: see text] IU, [Formula: see text], and [Formula: see text] IU, respectively. There was a significant negative correlation between the total FSH dosage and the cumulative live birth rate in women aged [Formula: see text] years ([Formula: see text], 95% confidence interval [−0.14, −0.08]; [Formula: see text]) and in those aged [Formula: see text] years ([Formula: see text], 95% confidence interval [−0.11, −0.02]; [Formula: see text]). Conclusions: The cumulative live birth rate decreased as the total FSH dose increased. These findings suggest that physicians may wish to avoid prescribing a high dose of FSH in women undergoing IVF/ICSI who are predicted not to have a poor response.

10252-COT-27 TWO CASES OF OVARIAN HYPERSTIMULATION SYNDROME IN A YOUNG FEMALE PATIENTS WITH GLIOMA WHO UNDERWENT OOCYTE CRYOPRESERVATION.

Abstract Young women with malignant gliomas may wish to have their oocytes preserved prior to the start of chemotherapy in order to maintain fertility. In such cases, fertility drugs make it possible to mature and retrieve multiple oocytes in the ovaries. However, the ovaries may overreact and mature many oocytes at once, resulting in adverse symptoms such as abnormal swelling of the ovaries, abdominal discomfort and pain, nausea and vomiting, abdominal distention, fluid retention (edema), and difficulty breathing. This is called ovarian hyperstimulation syndrome (OHSS), in which excessive secretion of estradiol associated with multiple follicle development leads to excessive production of VEGF, which rapidly increases the vascular permeability of systemic organs, resulting in a fluid shift into the third space, which is thought to cause ascites and pulmonary edema. In severe cases, intravascular dehydration and decreased circulating plasma volume can cause hypotension and oliguria, and the increased coagulation capacity associated with hemoconcentration can cause serious thrombotic complications. The severity of OHSS is classified into three levels: mild, moderate, and severe, based on four findings: subjective symptoms, the amount of pleural effusion, the degree of ovarian enlargement (ovarian diameter), and blood test findings. Early detection and appropriate management are essential. We have had two cases of OHSS, one of which was moderate and resolved successfully, while the other became severe, and we had to postpone the start of radiotherapy for a week longer than planned. Severe OHSS is a medical complication that neurosurgeons rarely encounter, and we report this case with a literature review.

#52 : Cumulative Live Birth Rates (CLBR) According to the Number of Oocytes Retrieved in Minimal Stimulation In Vitro Fertilization (IVF) Treatment

Background and Aims: Ovarian stimulation is necessary for IVF in order to stimulate the development of multiple follicles in a single cycle. To optimize ovarian stimulation, there is a need to know the optimum number of oocytes retrieved to achieve a live birth. This retrospective study aimed to evaluate the relationship between the number of oocytes retrieved and the cumulative live birth rates among patients undergoing mild stimulation IVF treatment in a private fertility clinic in the Philippines. Method: Ovulation was induced using clomiphene citrate in combination with lower doses of gonadotropins. Final oocyte maturation was triggered with hCG or GnRHa. Mature eggs were fertilized using intracytoplasmic sperm injection. Only 1 to 2 embryos were transferred per cycle. Results: A total of 1,431 patients that had 1,817 fresh and frozen transfer cycles were included. Average age of women was 35.70 ± 3.97 (min. 20, max. 46). Binary logistic regression shows that age is a predictor of live birth, with a critical value of 34.6 years. CLBR was estimated using Kaplan-Meier survival analysis, with the number of oocytes as the time factor. The estimated mean number of oocytes retrieved for cases that had live birth was 8.90 ± 0.30. Half of the cases that had live birth had an estimated oocyte number of 7.0 ± 0.194. Live birth steadily increased with the number of oocytes but declined beyond 25 oocytes. Retrieval of 6 to 9 oocytes was enough to achieve 40-60% CLBR. In comparison, conventional IVF recommends 8 to 15 oocytes to achieve similar CLBR. Conclusion: Retrieval of lower numbers of oocytes with minimal stimulation IVF can produce desirable success rates comparable to rates achieved with higher numbers of retrieved oocytes in conventional IVF.

P-652 RCT to evaluate the necessity of routine luteal phase support after ovarian stimulation by oral ovulogens in intrauterine insemination (IUI) cycles

Abstract Study question 1. Is it beneficial to provide luteal phase support in each IUI cycle stimulated by oral ovulogens ? Summary answer 1. Luteal phase support with progesterone makes no significant difference in clinical pregnancy rate in oral ovulogen stimulated IUI cycles. What is known already ART (assisted reproductive techniques) involves superovulation to achieve the development of multiple follicles. This produces supraphysiological E2 levels that causes feedback inhibition of FSH and LH in luteal phase leading to luteal phase defect. In IUI,only few follicles develop ,so the above phenomenon is rarely seen. Today widespread use of progesterone in clinical practice has become a habit, instead of the need, adding to burden of medication and cost to the patient without much evidence to recommend it. Study design, size, duration Prospective Randomized control over 1 year 200 IUI cycles randomized to either supplementing luteal phase with tab dydrogesterone (Group A ,no.100) or not supplementing (Group B, No. 100) as per inclusion(Unexplained infertility,Mild male factor,Donor sperm IUI, PCOD,Coital factors,Mild endometriosis) and exclusion criteria(IUI with gonatotropin, age more than 38 years, thin endometrium ,previous two or more IUI failures, history suggestive of luteal phase defect, recurrent pregnancy loss, structural uterine anomaly, history of endocrine or autoimmune diseases) Participants/materials, setting, methods Transvaginal scan was conducted on day 2 , followed by ovarian stimulation with letrozole 2.5 mg for 5 days. HCG trigger (10000IU) was given When follicle reached 18- 24mm with endometrium >7mm and IUI timed 36 hours after. Computer generated random allocation was done with one group receiving luteal phase support with tab dydrogesterone 10mg twice a day(Group A)and other not receiving LPS(Group B).Outcome of study was studied in terms of clinical pregnancy on USG. Main results and the role of chance Among the patients recruited, infertility was observed to be primary infertility in 74% cases in Group B and 72% in Group A , whereas secondary infertility was observed in 26% cases in group B and 28% in group A (P = 0.75). Group B included mild male factor in 29%, 24% of polycystic ovarian syndrome, 17 % tubal, 15% poor ovarian reserve, 9% of unexplained and 6% had endometriosis. Whereas in group A, PCOS (25%),male factor (21%),poor ovarian reserve and unexplained in 20%, endometriosis in 8% and 6% had tubal infertility. Among patients, mean endometrium thickness on trigger day of group Band group A were 8.46 ± 1.36mm and 8.44 ± 1.17mm respectively. Conception was reported by a positive urine pregnancy test . Among Group B , 23% had a positive urine pregnancy test. Whereas, in controls Group A 21% had a positive test, the difference being statistically non-significant (p value = 0.733) Outcomes were expressed terms of clinical pregnancy described as the presence of intrauterine sac with foetal heart rate. Clinical pregnancy rate was 22% of group B and 21% in group A , difference was statistically nonsignificant (p = 0.755). Limitations, reasons for caution Due to limited period of study and hence restricted sample size, the trial needs to be extended over a larger sample size for further interpretation. Further subgroup analysis needs to be conducted when a larger study group is recruited. Wider implications of the findings This study suggested that luteal support does not significantly affect the clinical pregnancy rate in IUI cycles with oral ovulogens .Our study could be a basis for further analysis to determine whether treatment burden in the form of luteal phase support can be reduced in such patients. Trial registration number not applicable

Recombinant equine FSH superovulation in the mare

Inducing ovulation of multiple follicles has been an elusive goal in equine reproduction. Administration of porcine FSH, ovine FSH or GnRH agonists only yield a marginal increase in ovulation rate in mares. Early studies utilizing single chain recombinant equine FSH (reFSH) reported stimulation of follicular development in anestrous mares and increased ovulation rates in cycling mares. Unfortunately, the original reFSH product from AspenBio Pharma, Inc. (Castle Rock, CO USA) is no longer in production. The goal of the current study was to evaluate the efficacy of a new single chain recombinant equine FSH (reFSH-FP; FertilPlus Partners, LLC; Conroe, TX USA) in stimulation of follicular development in mares in two pilot studies as compared to the original reFSH (reFSH-AB; AspenBio Pharma). Study 1. Nine seasonally anestrous mares (follicle diameter 19 ± 3.7 mm) were randomly assigned to one of 3 treatment groups (n=3/group): saline placebo, reFSH-AB (0.65 mg, IM, q12), reFSH-FP (0.65 mg, IM, q 12h) for a maximum of 7 days. A dose of hCG (2,500 units, IV) was administered after a 36-hour coast period once one or more follicles attained a diameter of ≥ 35 mm. The ovulation rates for the three treatment groups were 0, 10.3 ± 3.5, and 6.0 ± 2.0 ovulations for the saline placebo, reFSH-AB and reFSH-FP, respectively. Study 2. Twelve normally cycling mares were randomly assigned to one ofthe same treatment protocols as described for Study 1 above (n=4 per group). All mares were in mid-diestrus at the onset of treatment and a dose of cloprostenol (250 µg, IM) was administered on the second day of treatment. Mares were treated for a maximum of 7 days. HCG (2,500 units, IV) was administered after a coast period of 36 hours when one or more follicles attained a diameter of ≥ 35 mm. Ovulation rates were 1.3 ± 0.5, 5.5 ± 5.3, and 5.3 ± 1.0 ovulations for the saline placebo, reFSH-AB and reFSH-FP, respectively. All mares ovulated in the placebo and reFSH-FP groups after administration of hCG; one mare in the reFSH-AB group developed 12 follicles > 35 mm in diameter, but failed to ovulate after administration of hCG. Collectively, these studies confirm that the new recombinant equine FSH product (reFSH-FB; FertilPlus) has biological activity in the mare similar to that of the original reFSH product (reFSH-AB; AspenBio Pharma) and is capable of stimulating development of multiple large follicles in deep anestrus and cycling mares. However, significant hurdles remain before a recombinant equine FSH product could be available for commercial production, distribution and clinical use. Additional research will be needed to evaluate safety, dosage, frequency of administration, timing of administration of an ovulation induction agent, and fertility following reFSH treatment.

Plane of nutrition and FSH-induced superovulation affect the expression of steroid hormone receptors and growth factors in caruncular tissue of non-pregnant sheep

Implantation is a critical step in the establishment of pregnancy and an important part of embryo-maternal contact. Uterine receptivity can be affected by changes in body condition and the maternal endocrine milieu, including those caused by the use of exogenous gonadotropins in controlled ovarian hyperstimulation to induce the development of multiple follicles. This study demonstrates the effects of FSH-mediated ovarian hyperstimulation on the caruncles of ewes under various feeding regimes. Sheep were classified into 3 categories: control fed (CF), overfed (OF), or underfed (UF). In each group, animals were superovulated with FSH or injected with a saline solution (non-treated control). Uterine caruncles were collected at the early (d 5) and mid-luteal phase (d 10) of the estrous cycle. The transcript levels of steroid hormone receptors (ESR1, ESR2, PGR) and growth factors (IGF1, IGF2, VEGFA) were investigated and their expression localized by immunohistochemical staining. As for the main findings, day of the estrous cycle affected expression of ESR1, IGF1 and IGF2, but not of ESR2, PGR and VEGFA; both feeding and superovulation had modulatory effects, with feeding (UF/OF) stimulating expression of all genes studied, and superovulation altering expression of some genes, eg IGF1, PGR and ESR1 and ESR2, in CF animals. Similarly, feeding (UF/OF) altered responsiveness to superovulation for PGR on d 5 and ESR1/ESR2 on d 5 and/or 10. Our data emphasize possible effects of dietary and/or hormonal stimuli on uterine physiology, which may affect pregnancy outcomes by disrupting uterine functionality.

P–465 Effect of letrozole over Ki–67 expression in breast cancer during controlled ovarian stimulation (COS) for fertility preservation (FP): case report

Abstract Study question Does concomitant letrozole administration during COS alter Ki–67 expression in women undergoing FP procedures before breast cancer surgery? Summary answer Concomitant letrozole administration during COS, even for a short period, can reduce Ki–67 expression in breast cancer. What is known already The biggest concern with COS in breast cancer patients is the increase in serum estradiol levels, caused by the development of multiple follicles simultaneously. This has always been a major hindrance to the use of traditional ovarian stimulation regimens in these patients, due to the large amount of evidence on the pathogenetic role of estrogen in breast cancer propagation. To limit the rise of estradiol during COS, most centers have adopted concomitant letrozole administration. Recently, some studies have reported changes in tumor pathology after letrozole administration, such as a significant fall in Ki–67 expression. Study design, size, duration Case report including 2 patients undergoing COS with concomitant letrozole administration for 12 days before breast cancer surgery. Participants/materials, setting, methods The first patient was a 28-year-old Caucasian woman with a breast biopsy showing an infiltrating ductal carcinoma in the upper external quadrant of the right breast. The second patient was a 33-year-old Caucasian woman with a diagnosis of infiltrating ductal carcinoma of the upper external quadrant of the left breast. Both patients underwent COS with concomitant letrozole administration 5 mg daily for 12 days. Ovarian stimulation was performed using a GnRH-antagonist random-start protocol. Main results and the role of chance In the first patient, Ki–67 expression in the initial biopsy was 55%. After completion of FP procedures, she underwent quadrantectomy with sentinel-lymphnode biopsy. In the final histopathological report Ki–67 expression fell to 25%. In the second patient, the first biopsy showed a Ki–67 expression of 30%, while after mastectomy it fell to 10%. Limitations, reasons for caution Only 2 patients were included in the study. Wider implications of the findings: COS is feasible before breast cancer surgery, as long as an adequate cancer biopsy with immunohistochemical evaluation has been collected. Cytological diagnosis is not enough to start FP procedures. Evaluation of biological parameters after letrozole administration could lead to underestimation of cancer proliferation rate and to inappropriate treatment strategies. Trial registration number NA

Metabolomics analysis of follicular fluid coupled with oocyte aspiration reveals importance of glucocorticoids in primate periovulatory follicle competency

Gonadotropin administration during infertility treatment stimulates the growth and development of multiple ovarian follicles, yielding heterogeneous oocytes with variable capacity for fertilization, cleavage, and blastocyst formation. To determine how the intrafollicular environment affects oocyte competency, 74 individual rhesus macaque follicles were aspirated and the corresponding oocytes classified as failed to cleave, cleaved but arrested prior to blastulation, or those that formed blastocysts following in vitro fertilization. Metabolomics analysis of the follicular fluid (FF) identified 60 unique metabolites that were significantly different between embryo classifications, of which a notable increase in the intrafollicular ratio of cortisol to cortisone was observed in the blastocyst group. Immunolocalization of the glucocorticoid receptor (GR, NR3C1) revealed translocation from the cytoplasm to nucleus with oocyte maturation in vitro and, correlation to intrafollicular expression of the 11-hydroxy steroid dehydrogenases that interconvert these glucocorticoids was detected upon an ovulatory stimulus in vivo. While NR3C1 knockdown in oocytes had no effect on their maturation or fertilization, expansion of the associated cumulus granulosa cells was inhibited. Our findings indicate an important role for NR3C1 in the regulation of follicular processes via paracrine signaling. Further studies are required to define the means through which the FF cortisol:cortisone ratio determines oocyte competency.

Placentation in the African Elephant (Loxodonta africana).

The female elephant shows a 3-week "follicular phase" to commence her 16-week estrous cycle at the end of which a second surge in pituitary luteinizing hormone (LH) release matures and ovulates an ovarian follicle in association with estrous behavior and mating, whereas the first LH surge at the start of the follicular phase causes luteinization of 3-5 partially developed follicles. The prolonged pregnancy of 22months is supported by a zonary endotheliochorial placenta which secretes placental lactogen (ePL) from around 40days of gestation in association with replacement of the lumenal epithelium of the endometrium by trophoblast and the development of large corpora lutea (CLs) in the maternal ovaries from the previously formed luteinized follicles in response to the first LH peak early in the follicular phase. The zonary placenta develops above, rather than within, the endometrium. The elephant placenta secretes neither estrogens nor progestagens throughout gestation, as pregnancy maintenance relies on 5α-dihyroprogesterone and other 5α reduced progestagens secreted by secondary CLs stimulated by ePL and the stromal tissue of the fetal gonads, which become extremely enlarged during the second half of the 22-month pregnancy. In female fetuses, this ovarian enlargement includes the development and subsequent regression of multiple primary and secondary follicles with a consequent substantial decline in primary follicle numbers at birth. During the next 8-9years of pre-pubertal life, however, oocyte and primary follicle numbers recover to levels near those found in late gestation, which may be evidence of postnatal oogenesis occurring in the elephant.

Embryonic response to high beta-hydroxybutyrate (BHB) levels in postpartum dairy cows

Cows at the beginning of lactation often do not meet their energy needs by feeding and therefore mobilize body fat, which produces ketone bodies, including β-hydroxybutyrate (BHB). They are nevertheless usually inseminated around 60 d postpartum, when they are still in this characteristic period of energy deficit. The aim of this study was to observe the effects of negative energy balance on embryo quality and to identify ways to improve the fertility of dairy cows. Holstein cows (n = 18) grouped as high or low BHB based on blood measurement at day 45 postpartum were estrus-synchronized and treated with follicle-stimulating hormone to obtain multiple follicle development, induced to ovulate and inseminated with sexed semen around day 60 postpartum. Of the 290 embryos collected over 16 mo, 159 were of quality I to IV. Based on microarray analysis of gene expression, exposure to an energy deficit metabolic environment (high BHB) during early development appeared to modify signaling by the mTOR and sirtuins pathways in the embryo, implying mitochondrial dysfunction and inhibition of transcription, leading to slower cell division, thus programming the embryo to be more energy efficient. Altered methylation markers suggested that such coping mechanisms might persist into adulthood.

Effects of controlled ovarian hyperstimulation protocols on uterine markers

Controlled ovarian hyperstimulation is based on the development of multiple follicles in the same cycle as achieving the optimal number and quality of oocytes and is a common practice in infertility treatment. In this application, clomiphene citrate and gonadotropins are the most commonly used agents. These agents have some effects on reproductive tissues. Uterine tissue is susceptible to hormonal changes and undergoes a change in hormone action in terms of protein profile, secretory content and molecular structure during the menstrual cycle. In this review, it is aimed that controlled ovarian hyperstimulation has effect on female reproductive organs with different markers and methods, how to gather information about where the change occurs due to the functions of these markers, and to present this information to scientists working in the field by being related to each other. It is seen that controlled ovarian hyperstimulation may have negative effects on many functions such as uterine growth, receptivity, and altered expressions of the markers. Articles in this review, it is generally recognized that the expression of a majority of receptive proteins is decreased, which is thought to have an adverse effect on live birth rates and infertility treatment. More clinical trials are needed for these markers in order to increase live birth rates, implant success and to develop infertility treatment.

Dose and administration protocol for FSH used for ovarian stimulation affect gene expression in sheep cumulus-oocyte complexes.

The present study evaluated the effect of four ovarian stimulation protocols on the follicular population and molecular status of cumulus-oocyte complexes (COCs). Twelve Santa Inês ewes (in a cross-over design) received 80 or 120mg FSH alone in a multiple-dose (MD80 and MD120) regimen or in combination with 300IU equine chorionic gonadotrophin (eCG) in a one-shot (OS80 and OS120) protocol. The follicular population, COC recovery rate, mean COCs per ewe and the rate of brilliant Cresyl blue-positive (BCB+) COCs were similar among treatments (P>0.05). The expression of markers of oocyte competence (ZAR1, zygote arrest 1; MATER, maternal antigen that embryo requires; GDF9, growth differentiation factor 9; BMP15, bone morphogenetic protein 15; Bcl-2, B-cell lymphoma 2; BAX, Bcl-2 associated X protein) and the steroidogenic pathway (ERα, oestrogen receptor α; LHr, LH receptor; FSHr, FSH receptor; STAR, steroidogenic acute regulatory protein) was affected by stimulation. Specifically, the expression of markers of the steroidogenic pathway was reduced with increasing FSH dose in the OS protocol. FSH at a dose of 80mg reduced the expression of FSHr and ERα in the OS versus MD protocol. Conversely, in MD protocol, only LHr was affected by increasing FSH dose. In conclusion, 80mg FSH in the MD or OS protocol was sufficient to promote the development of multiple follicles and obtain fully grown (BCB+) oocytes. The MD protocol may be more appropriate for the production of better-quality oocytes.

Corpora lutea in superovulated ewes fed different planes of nutrition

The corpus luteum (CL) is an ovarian structure which is critical for the maintenance of reproductive cyclicity and pregnancy support. Diet and/or diet components may affect some luteal functions. FSH is widely used to induce multiple follicle development and superovulation. We hypothesized that FSH would affect luteal function in ewes fed different nutritional planes. Therefore, the aim of this study was to determine if FSH-treatment affects (1) ovulation rate; (2) CL weight; (3) cell proliferation; (4) vascularity; (5) expression of endothelial nitric oxide (eNOS) and soluble guanylate cyclase (sGC) proteins; and (6) luteal and serum progesterone (P4) concentration in control (C), overfed (O), and underfed (U) ewes at the early- and mid-luteal phases. In addition, data generated from this study were compared to data obtained from nonsuperovulated sheep and described by Bass et al. Ewes were categorized by weight and randomly assigned into nutrition groups: C (2.14 Mcal/kg; n = 11), O (2xC; n = 12), and U (0.6xC; n = 11). Nutritional treatment was initiated 60 d prior to day 0 of the estrous cycle. Ewes were injected with FSH on day 13–15 of the first estrous cycle, and blood samples and ovaries were collected at early- and mid-luteal phases of the second estrous cycle. The number of CL/ewe was determined, and CL was dissected and weighed. CL was fixed for evaluation of expression of Ki67 (a proliferating cell marker), CD31 (an endothelial cell marker), and eNOS and sGC proteins using immunohistochemistry and image analysis. From day 0 until tissue collection, C maintained, O gained, and U lost body weight. The CL number was greater (P < 0.03) in C and O than U. Weights of CL, cell proliferation, vascularity, and eNOS but not sGC expression were greater (P < 0.001), and serum, but not luteal tissue, P4 concentrations tended to be greater (P = 0.09) at the early- than mid-luteal phase. Comparisons of CL measurements demonstrated greater (P < 0.01) cell proliferation and serum P4 concentration, but less vascularity at the early and mid-luteal phases, and less CL weight at the mid-luteal phase in superovulated than nonsuperovulated ewes; however, concentration of P4 in luteal tissues was similar in both groups. Thus, in superovulated ewes, luteal cell proliferation and vascularity, expression of eNOS, and serum P4 concentration depend on the stage of luteal development, but not diet. Comparison to control ewes demonstrated several differences and some similarities in luteal functions after FSH-induced superovulation.

Dynamics of the development of multiple follicles by early versus late hCG administration in ART program

Objective: To evaluate, in patients stimulated with recombinant FSH and GnRH antagonists, whether triggering the final maturation of oocytes affects IVF outcomes.Study design: Five hundred and six IVF procedures were divided into three groups according to the timing of hCG administration: when at least 2 follicles reached the diameter of 17 mm, at least 2 follicles reached 18 mm and at least 2 follicles reached 20 mm. The main outcome was the number of mature oocyte that was the dependent variable of a multivariate model whose independents were, age, AFC, hCG timing, E2 levels at hCG day, number of follicles in different categories of dimension. Secondary endpoints were to compare fertilization, implantation and pregnancy rates in a multilevel multivariate model whose covariates were age, BMI, AFC, embryo quality and cause of infertility.Results: Timing did not result a statistically significant factor influencing the number of oocytes collected, which was influenced by age, AFC, number of follicles between 12.1 and 15.9 mm and E2 levels. Implantation rate and pregnancy rate appear to be affected only by embryo quality.Conclusion: The number of oocytes collected and the probability of pregnancy are not associated with the time of hCG administration.

Uterine focal adhesions are retained at implantation after rat ovarian hyperstimulation.

Controlled ovarian hyperstimulation is an essential component of IVF techniques to ensure proliferation and development of multiple ovarian follicles, but the effects of these hormones on the endometrium are largely unknown. During normal pregnancy in rats, there are significant changes in the basal plasma membrane of uterine epithelial cells (UECs) at the time of receptivity, including loss of focal adhesions. This enables the UECs to be removed from the implantation chamber surrounding the blastocyst, thus allowing invasion into the underlying stroma. This study investigated the influence of ovarian hyperstimulation (OH) on the basal plasma membrane of UECs during early pregnancy in the rat. Immunofluorescence results demonstrate the presence of paxillin, talin, integrin β1 and phosphorylated FAK (Y397FAK) in the basal portion of UECs at the time of implantation in OH pregnancy. TEM analysis demonstrated a flattened basal lamina and the presence of focal adhesions on the basal surface at this time in OH pregnancy. Significantly low full-length paxillin, high paxillin δ and integrin β1 were seen at the time of implantation in OH compared with those in normal pregnancy. The increase in paxillin δ suggests that these cells are less mobile, whereas the increase in integrin β1 and Y397FAK suggests the retention of a stable FA complex. Taken together with the increase in morphological focal adhesions, this represents a cell type that is stable and less easily removed for blastocyst implantation. This may be one mechanism explaining lower implantation rates after fresh embryo transfers compared with frozen cycles.

Natural ovarian stimulation (NATOS) successfully combines low estradiol levels and multiple follicle development and may be suitable for fertility preservation in women with estrogen-dependent cancers

Supraphysiologic E2 levels are unwelcome in fertility preservation candidates with breast cancer. Aromatase inhibitors have been used during controlled ovarian hyperstimulation (COH) to avoid hyper-estrogenism but serum oestrogens levels can exceed 600pg/mL when numerous oocytes are recovered. Therefore, we tested an innovative COH protocol that virtually curtailed endogenous LH levels by using strong and sustained GnRH antagonist doses while maintaining standard exogenous FSH-only administration.

Effect of single-chain ovine gonadotropins with dual activity on ovarian function in sheep.

We examined the half-life and biological activity of two single-chain proteins that combined portions of ovine FSH and LH. We proposed the hypothesis that these chimeric proteins would display LH and FSH activities and would promote follicle maturation in ewes. Estrus activity was synchronized using progestogen-impregnated vaginal pessaries. To negate the impact of endogenous LH and FSH, animals received serum-containing antibodies against GNRH 1 day before pessary removal (PR). At PR sheep (five animals per group) received a single injection (10 IU/kg, i.v.) of either the ovine-based (oFcLcα) gonadotropin analog, an ovine-based analog containing oLHβ truncated at the carboxyl terminus (oFcL(ΔT)cα), or a human-based gonadotropin analog (hFcLcα). Control animals received a comparable amount of gonadotropin-free protein. Ovulation was induced 3 days after PR using human chorionic gonadotropin (1000 IU, i.v.). Ovaries were collected 11 days after PR. Neither estradiol (E2) or progesterone (P4) production, development of preovulatory follicles or corpora lutea (CL) were noted in control animals receiving gonadotropin-free protein. Significant increase in the synthesis of E2 and P4 was noted in sheep receiving the dually active gonadotropin analogs. The number of CLs present 11 days after PR was significantly increased in sheep receiving the chimeric glycoproteins compared with control animals. The magnitude of the secretory and ovarian responses did not differ between hFcLcα and oFcLcα or between oFcLcα and oFcL(ΔT)cα. Immunoactivity of LH and FSH was low in control animals, but was significantly elevated in sheep receiving the gonadotropin analogs. In conclusion, ovine-based gonadotropin analogs are functionally active in sheep and a single injection is adequate to induce the development of multiple ovulatory follicles.

Optimal control for predicting customized drug dosage for superovulation stage of in vitro fertilization

in vitro fertilization (IVF) is one of the most highly pursued assisted reproductive technologies (ART) worldwide. IVF procedure is divided into four stages: Superovulation, Egg-retrieval, Insemination/Fertilization and Embryo transfer. Among these superovulation is the most crucial stage since it involves external injection of hormones to stimulate development and maturation of multiple follicles or oocytes. Although numerous advancements have been made in IVF procedures, little attention has been given to modifying the existing protocols based on a ‘patient specific’ predictive model. A model for follicle growth and number change as a function of the injected hormones and patient characteristics has been developed and validated for data available on 50 superovulation cycles. The model has 9 patient specific parameters which can be determined from the initial 2 days of observation and can help in projecting the superovulation outcome for the ongoing cycle. Based on this model, the dosage of the hormones to stimulate multiple ovulation or follicle growth is predicted by using the theory of optimal control. The objective of successful superovulation is to obtain maximum number of mature oocytes/follicles within a particular size range. Using the mathematical model of follicle growth dynamics and optimal control theory, optimal dose and frequency of medication customized for each patient (n=5) is predicted for obtaining the desired result. The results indicate a better final day follicle size distribution when the dosage of the hormones is varied by some amounts as compared to the actual dosage given to the patient in the existing cycles. This ensures a better success rate for the superovulation cycles and reduces the costs of excess medication and daily monitoring. The idea is to provide the medical practitioners with a guideline for planned treatment, for a procedure currently based on trial and error in order to get better success rates.

Impact Of Thrombin Generation, Tissue Factor Activity and Thrombomodulin Activity On The Positivity Of Assisted Reproductive Technique In Infertile Women

IntroductionWomen undergoing assisted reproductive techniques (ART) are given gonadotrophins to promote the development of multiple follicles within their ovaries. This treatment may be associated with a risk of ovarian hyperstimulation syndrome and venous or arterial thrombosis. The association of clinical and biological criteria of hypercoagulability might contribute to the identification of patients at risk and probably could predict success of ART.The aim of this study was to evaluate thrombin generation, thrombomodulin activity, tissue factor (TF) activity, and procoagulant phospholipids in selected women undergoing ART. We also assessed the potential correlation between the levels of the studied biomarkers and the outcome of the ART. Material and MethodA cohort of 27 infertile women eligible for ART and 30 healthy age matched women was studied. All patients were Caucasian aged 33.75 ± 3.52 years and weight 61.08 ± 8.10 kg. Women included in the study did not present any personal or family history of VTE or known thrombophilia. They did not present any signs of hemorrhagic syndrome and did not suffer from any known autoimmune disease. Blood samples were taken under fasting conditions at the following time-points: at the inclusion (T0), between the 5th and 8th day of ovarian stimulation with gonadotrophines (T1) and at the day of HCG administration (T2). Thrombin generation (TG) in plasma was assessed using the Calibrated Automated Thrombogram assay (using, PPP-Reagent-5pM TF from Diagnostica Stago, France), Plasma levels of thrombomodulin activity (TMa), and TF activity (TFa) were measured by home-made tests, Procoagulant phospholipids (PPL) dependent clotting time was measured using a factor Xa-based assay (STA-R Procoag-PPL, Diagnostica Stago, France). ResultsThe endogenous thrombin potential (ETP), PPL, TMa and TFa were significantly higher in the ART-T0 group as compared to the control group. At ART-T2 a significant increase of TG was observed as compared to ART-T0.At ART-T0 44.5%, 44.4 % and 33.3 % of women had ETP, TFa and TMa higher than the Upper Normal Limit respectively (UNL = mean+2 S.D.). Among with negative ART 89% and 91.7% showed TMa and TFa levels > UNL at ART-T0. At T1 50% of women had a least one parameter of TG higher than the UNL. At ART T2 65.2 % of women had TG > UNL. At the same time, 87.5% and 83.4% of women with negative ART had levels of TMa and TFa > UNL. ConclusionThis study analyzed the profile of thrombin generation in infertile women eligible for ART and investigated the influence of hormone treatment with gonadotropins and HCG on TG and levels of TMa and TFa. Hypercoagulability, in terms of increased ETP is present in 46% of infertile women eligible for ART. These women remain in a hypercoagulable state throughout the entire period of hormone treatment. The most significant finding of this study was that 33% of patients show a value superior to the UNL for thrombomodulin and 45% for tissue factor. Interestingly 89%of women with negative ART had TMa higher than the UNL. Respectively 91% of women with negative ART had TFa levels higher than the UNL Disclosures:No relevant conflicts of interest to declare.ControlART T0ART T1ART T2n=30n=27n=24n=23Thrombin generationETP (nM.min)1670 ± 2462105 ± 683***1812 ± 6021997 ± 574**Peak (nM)345 ± 61384 ± 96342 ± 108375 ± 85tt Peak (min.)5.29 ± 0.665.58 ± 1.10*5.31 ± 0.975.13 ± 1.03MRI (nM/min)150 ± 42147 ± 42136 ± 55157 ± 45Haemostasis factorsPPL (sec.)60.21 ± 14.3236.02 ± 5.72***37.90 ± 6.91***36.21 ± 6.30***TM (%)90 ± 18115 ± 47**117 ± 44***117 ± 35***TFa (pM)0.24 ± 0.110.46 ± 0.41**0.47 ± 0.37***0.34 ± 0.27(***<0.001; **<0.01; *<0.05 vs control)