Effect Of Ovarian Stimulation Research Articles

Effect of ovarian stimulation on developmental speed of preimplantation embryo in a mouse model.

Ovarian stimulation protocols are widely used to collect oocytes in assisted reproductive technologies (ARTs). Although the influence of ovarian stimulation on embryo quality has been described, this issue remains controversial. Here, we analyzed the influence of ovarian stimulation on developmental speed and chromosome segregation using live cell imaging. Female mice at the proestrus stage were separated by the appearance of the vagina as the non-stimulation (-) group, and other mice were administered pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) as the stimulation (+) groups. The cumulus-oocyte complexes from both groups were inseminated with sperm suspensions from the same male mice. Fertilization rates and developmental capacities were examined, and the developmental speed and frequency of chromosome segregation errors were measured by live-cell imaging using a Histone H2B-mCherry probe. The number of fertilized oocytes obtained was 1.4-fold more frequent in the Stimulation (+) group. The developmental rate and chromosome stability did not differ between the groups. Image analysis showed that the mean speed of development in the stimulation (+) group was slightly higher than that in the non-stimulation (-) group. This increase in speed seemed to arise from the slight shortening of the 2- and 4-cell stages and third division lengths and consequent synchronization of cleavage timing in each embryo, not from the emergence of an extremely rapidly developing subpopulation of embryos. In conclusion, ovarian stimulation does not necessarily affect embryo quality but rather increases the chances of obtaining high-quality oocytes in mice.

O-149 Endometrial thickness as a predictor of live birth and perinatal outcomes following oocyte donation

Abstract Study question Does endometrial thickness (EMT) predict the occurrence of live birth following oocyte donation? Summary answer Our study found that EMT is predictive of live birth in oocyte donation. What is known already EMT is a widely used surrogate marker of endometrial receptivity and is believed to be associated with in vitro fertilization pregnancy outcomes. However, the evidence thus far is mixed, with many studies (and even meta-analyses) suggesting that EMT may have little/no predictive value. One potential reason for the heterogeneity in results may be that most studies assessed mostly fresh autologous embryo transfers, without accounting for embryo quality nor the effect exogenous ovarian stimulation may have on endometrial receptivity and subsequent pregnancy/perinatal outcomes. Moreover, most subcategorized EMT to facilitate interpretation, which may have inadvertently introduced bias to the analyses. Study design, size, duration This retrospective, multicentre, cohort study analyzed all single blastocyst embryo transfers following oocyte donation from 2010-2019 subdivided into regular intervals of EMT (<7.0 mm, 7.0-8.9 mm, 9.0-10.9 mm, ≥11.0 mm). EMT was also evaluated as a continuous variable, comparing the best-fitting fractional polynomial against the linear function, to minimize bias due to residual confounding that may occur following the categorization of continuous variables. Confounder adjustment was performed using multivariable generalized estimating equations regression analysis. Participants/materials, setting, methods The oocyte donation model was chosen to minimize the influence of potential confounding factors such as poor embryo quality and the effect of ovarian stimulation on endometrial receptivity. The main objective of the study was to compare live birth rates (LBR). Our secondary outcomes included other surrogate pregnancy (hCG-positive pregnancy, clinical pregnancy, and miscarriage rates) and perinatal (gestational age at birth, preterm birth under 37 weeks, birthweight z-score, small and large for gestational age) outcomes. Main results and the role of chance In total, 33915 embryo transfers were analyzed. Confounder variables included were female donor and recipient age, recipient BMI, female factor infertility, male factor infertility, number of mature oocytes donated, oocyte status (fresh versus vitrified), sperm source (partner or donor), embryo status (fresh versus vitrified), embryo quality, endometrial preparation (natural or artificial cycle) and year of transfer. When compared to the reference EMT of 9.0-11.0 mm, EMT <7.0 mm was associated with lower hCG positive pregnancy rates (51.3% vs 57.6%; aOR 0.82 CI 0.75-0.89), lower clinical pregnancy (41.9% vs 49.1%; aOR 0.79 CI 0.73-0.86), higher miscarriage rates per hCG positive pregnancy (37.5% vs 31.4%; aOR 1.31 CI 1.16-1.47) and lower LBRs per transfer (32.1% vs 39.5%; aOR 0.76 CI 0.69-0.83). Regarding perinatal outcomes, we found significantly higher preterm delivery rates following an EMT <7.0 mm (14.1% vs 9.9%; aOR 1.54 CI 1.21-1.96) and higher large for gestational age rates associated with EMT ≥11.0 mm (16.4% vs 12.2%; aOR 1.40 CI 1.07-1.83). The fractional polynomial analysis revealed that the subcategorization of EMT caused clinically-relevant residual confounding. Specifically, LBRs assumed an approximate bell-shaped relationship with EMT, with LBR rates decreasing for both thinner EMTs (<7 mm subgroup) and thicker EMTs (≥11.0 mm subgroup). Limitations, reasons for caution The retrospective nature of the study and the inherent risk of bias related to unmeasured confounding may have impacted the results. Another potential limitation is the lack of perinatal outcome data due to loss of follow-up, which should be considered when interpreting these results. Wider implications of the findings Our results support the use of EMT as a predictor of live birth and as a marker of endometrial receptivity and competence. Moreover, it stresses the potential danger of subcategorizing all EMT <7.0 mm and >11.0 mm as equivalent, as the study found that these subgroups have varying LBR. Trial registration number not applicable

P-333 The influence of hormonal stimulation on uterine peristalsis measured by ultrasound speckle tracking in women with IVF/ICSI treatment compared to normal ovulating women

Abstract Study question To investigate possible effects of ovarian stimulation during IVF/ICSI on uterine peristalsis features using ultrasound speckle tracking compared to normal ovulating women. Summary answer Patients with a normal uterus treated with exogenous hormones during IVF/ICSI treatment show significantly increased contraction amplitude and power parameters compared to normal ovulating women. What is known already Uterine peristalsis plays a role in procreation. Subjective visual inspection of uterine peristalsis is extensively researched throughout the menstrual cycle. Studies on uterine peristalsis in IVF/ICSI patients show higher frequency of uterine contractions during the periovulatory phase. Recently, a new automated quantitative method was validated to objectively analyse uterine strain using speckle tracking. Besides objective insight in frequency, also information on amplitude, power and coordination of motion is available. The effect of exogenous hormones on uterine contraction features has not objectively been studied using this new method. Study design, size, duration This multicentre prospective observational cohort study was performed between 2020 and 2022 in the Catharina Hospital in Eindhoven, the Netherlands, Embryolab Fertility Centre in Thessaloniki, Greece and University of Naples, Federico II in Naples, Italy. 30 normal ovulating patients served as controls. The primary outcomes were the contraction features frequency, amplitude, direction, velocity, power and coordination in women undergoing IVF/ICSI on the day of follicle aspiration compared to controls on cycle day 9 – 14. Participants/materials, setting, methods 19 women undergoing IVF/ICSI were included regardless of the treatment cycle number and stimulation protocol. Indication for IVF/ICSI treatment was male subfertility (n = 8), idiopathic subfertility (n = 5), tubal factor (n = 2), primary ovarian insufficiency (n = 2) or combined (n = 2), all women had normal uteri on transvaginal ultrasound. The control group consisted of 30 healthy pre-menopausal women with normal uteri on ultrasound and spontaneous regular cycles. Main results and the role of chance Each patient underwent a 2.5 – 4 minute transvaginal ultrasound of the uterus in mid-sagittal plane. The various contraction features were extracted using a quantitative dedicated speckle tracking algorithm analysis studying motion of the subendometrial junctional zone. Mean age of IVF/ICSI patients was 32.7 years and mean BMI was 25.6. Mean age of healthy controls was 33.8 years, BMI was 23.4, and cycle length was 27.44 days. Characteristics of IVF/ICSI patients and healthy volunteers showed no statistically significant differences. Both groups predominantly consisted of nulliparous women. In IVF/ICSI patients the contraction amplitude was significantly higher compared to controls (0.062 vs 0.040; p < 0.001). The contraction power (combination of amplitude and frequency) appeared to be significantly higher in IVF/ICSI patients compared to controls (1528.18 vs 683.44; p < 0.001). No significant differences were found in contraction frequency and coordination. Even though the mean coordination did not show statistically significant differences, the values were less scattered in IVF/ICSI patients, with a much smaller range of coordination values (1.04 – 3.06) compared to healthy volunteers (1.02 – 50.33). Limitations, reasons for caution The reported difference between IVF/ICSI patients and controls in contraction amplitude and power may not solely be attributed to the administration of exogenous hormones. The difference may also be due to an underlying cause for infertility in these patients. Another limitation of this study is the small sample size. Wider implications of the findings Future research should explore which uterine contraction profile is associated with viable pregnancy: during the natural menstrual cycle, or when treated with hormones. The question arises whether it is preferred to transfer the embryo in the stimulated cycle or implement a freeze-all policy and transfer during the natural menstrual cycle. Trial registration number NCT02310802

P–751 Immediate versus postponed frozen-thawed embryo transfer after IVF/ICSI: a systematic review and meta-analysis

Abstract Study question Can frozen embryo transfer (FET) be offered immediately after a stimulated IVF/ICSI cycle without compromising live birth rate (LBR)? Summary answer FET in the menstrual cycle immediately following the stimulated IVF/ICSI cycle was associated with a slightly higher LBR compared to standard postponed FET. What is known already It is standard clinical practice to postpone FET for at least one menstrual cycle following a failed fresh transfer or a freeze-all cycle. This practice is thought to minimize any possible residual negative effect of ovarian stimulation, with excessive steroid levels and multiple corpora lutea, on the resumption of a normal ovulatory cycle and receptivity of the endometrium. Even so, elective deferral of FET is an empirical strategy based on suggestions rather than solid scientific evidence and may unnecessarily delay time to pregnancy, causing frustration and decreased quality of life to couples. Study design, size, duration Systematic review and meta-analysis according to PRISMA guidelines. Original studies on subfertile women aged 18–46 with any indication for treatment with IVF/ICSI investigating the timing of FET after IVF/ICSI were included. Intervention was defined as FET in the menstrual cycle immediately following the stimulated IVF/ICSI cycle. Comparator was defined as FET in the second or subsequent menstrual cycle following IVF/ICSI. Risk of bias was assessed using Robins-I and quality of evidence using GRADE. Participants/materials, setting, methods PubMed (MEDLINE) and EMBASE databases were searched for MeSH and Emtree terms, as well as text words related to timing of FET, up to March 2020. There were no limitations regarding year of publication or duration of follow-up but to English language. The primary outcome was LBR. Secondary outcomes were implantation rate, pregnancy rate, clinical pregnancy rate (CPR), time-to-pregnancy, miscarriage rate (MR), cycle cancellation rate and patient wellbeing. Main results and the role of chance Out of 4124 search results, 15 studies were included in the review. Studies reporting adjusted odds ratios (aOR) for LBR, CPR and MR were included in meta-analyses. All studies (n = 15) were retrospective cohort studies involving a total of 6,304 immediate FET cycles and 13,851 postponed FET cycles including 8,019 matched controls. Twelve studies of very low to moderate quality reported no difference in LBR with immediate versus postponed FET. Two studies of moderate quality reported a statistically significant increase in LBR with immediate FET and one small study of very low quality reported better LBR with postponed FET. Trends in rates of secondary outcomes followed trends in LBR regarding timing of FET. The meta-analyses showed a significant advantage of immediate FET (n = 2,076) compared to postponed FET (n = 3,833), with a pooled aOR of 1.20 (95% CI 1.01–1.44) for LBR and a pooled aOR of 1.22 (95% CI 1.07–1.39) for CPR. Limitations, reasons for caution: Limitations include the retrospective design and heterogeneity of studies included, limiting comparison and pooling of data. With little transparency regarding cancellation rates, the risk of selection bias is apparent. Further, confounding by embryo quality is a limitation. Small sample sizes are a limitation to subgroup meta-analyses. Wider implications of the findings: The standard clinical practice of postponing FET for at least one menstrual cycle following a failed fresh transfer or a freeze-all cycle may not be best clinical practice. Randomized controlled trials including data on cancellation rates are highly needed to provide high grade evidence regarding clinical practice and patient counseling. Trial registration number Not applicable

P-751 Immediate versus postponed frozen-thawed embryo transfer after IVF/ICSI: a systematic review and meta-analysis

Abstract Study question Can frozen embryo transfer (FET) be offered immediately after a stimulated IVF/ICSI cycle without compromising live birth rate (LBR)? Summary answer FET in the menstrual cycle immediately following the stimulated IVF/ICSI cycle was associated with a slightly higher LBR compared to standard postponed FET. What is known already It is standard clinical practice to postpone FET for at least one menstrual cycle following a failed fresh transfer or a freeze-all cycle. This practice is thought to minimize any possible residual negative effect of ovarian stimulation, with excessive steroid levels and multiple corpora lutea, on the resumption of a normal ovulatory cycle and receptivity of the endometrium. Even so, elective deferral of FET is an empirical strategy based on suggestions rather than solid scientific evidence and may unnecessarily delay time to pregnancy, causing frustration and decreased quality of life to couples. Study design, size, duration Systematic review and meta-analysis according to PRISMA guidelines. Original studies on subfertile women aged 18-46 with any indication for treatment with IVF/ICSI investigating the timing of FET after IVF/ICSI were included. Intervention was defined as FET in the menstrual cycle immediately following the stimulated IVF/ICSI cycle. Comparator was defined as FET in the second or subsequent menstrual cycle following IVF/ICSI. Risk of bias was assessed using Robins-I and quality of evidence using GRADE. Participants/materials, setting, methods PubMed (MEDLINE) and EMBASE databases were searched for MeSH and Emtree terms, as well as text words related to timing of FET, up to March 2020. There were no limitations regarding year of publication or duration of follow-up but to English language. The primary outcome was LBR. Secondary outcomes were implantation rate, pregnancy rate, clinical pregnancy rate (CPR), time-to-pregnancy, miscarriage rate (MR), cycle cancellation rate and patient wellbeing. Main results and the role of chance Out of 4124 search results, 15 studies were included in the review. Studies reporting adjusted odds ratios (aOR) for LBR, CPR and MR were included in meta-analyses. All studies (n = 15) were retrospective cohort studies involving a total of 6,304 immediate FET cycles and 13,851 postponed FET cycles including 8,019 matched controls. Twelve studies of very low to moderate quality reported no difference in LBR with immediate versus postponed FET. Two studies of moderate quality reported a statistically significant increase in LBR with immediate FET and one small study of very low quality reported better LBR with postponed FET. Trends in rates of secondary outcomes followed trends in LBR regarding timing of FET. The meta-analyses showed a significant advantage of immediate FET (n = 2,076) compared to postponed FET (n = 3,833), with a pooled aOR of 1.20 (95% CI 1.01-1.44) for LBR and a pooled aOR of 1.22 (95% CI 1.07-1.39) for CPR. Limitations, reasons for caution Limitations include the retrospective design and heterogeneity of studies included, limiting comparison and pooling of data. With little transparency regarding cancellation rates, the risk of selection bias is apparent. Further, confounding by embryo quality is a limitation. Small sample sizes are a limitation to subgroup meta-analyses. Wider implications of the findings The standard clinical practice of postponing FET for at least one menstrual cycle following a failed fresh transfer or a freeze-all cycle may not be best clinical practice. Randomized controlled trials including data on cancellation rates are highly needed to provide high grade evidence regarding clinical practice and patient counseling. Trial registration number not applicable

Effect of ovarian stimulation on embryo aneuploidy and mosaicism rate

ABSTRACT There is a high incidence of chromosome abnormalities in human embryos that leads to a failed IVF cycle. Different studies have shown that maternal age is the determining factor in the appearance of chromosomal alterations in the embryo. However, the possible influence of ovarian stimulation on oocyte and embryo aneuploidies and mosaicism is controversial. A retrospective study was carried out in which 835 embryos from 280 couples undergoing reproductive treatment using their oocytes were chromosomally analyzed. A binary logistic regression analysis was performed to evaluate the relationship between different parameters characterizing controlled ovarian stimulation (COS) and the rate of aneuploidy and embryonic mosaicism. The embryo aneuploidy rate showed no association with the use of oral contraceptives, type, total and daily doses of gonadotropins, stimulation protocol type, and drugs used for ovulation trigger (p > 0.05). In contrast, the duration of the ovarian stimulation treatment was correlated with the aneuploidy rate: patients requiring more days of stimulation presented a lower rate of aneuploid embryos (p = 0.015). None of the variables studied showed any association with the rate of embryo mosaicism. However, the duration of COS showed association with the appearance of aneuploidy, suggesting that faster recruitment could be deleterious for those reassuming meiosis, yielding more abnormal karyotype. Abbreviations: IVF: in vitro fertilization; COS: controlled ovarian stimulation; PGT-A: preimplantation genetic test for aneuploidy; hCG: human chorionic gonadotropin; GnRH: gonadotropin-releasing hormone; LH: luteinizing hormone; FSH: follicle-stimulating hormone; NGS: next-generation sequencing; a-CGH: comparative genomic hybridization; TUNEL: Terminal transferase dUTP Nick End Labeling; FISH: fluorescent in situ hybridization

Effect of ovarian stimulation by different gonadotrophin treatments on in vivo and in vitro reproductive efficiency of rabbit does under high ambient temperature.

Non-lactating multiparous NZW rabbit does (n = 227) were used in two experiments. In the 1st experiment (n = 87), does were i.m. injected with 0.1-ml saline/doe in day 0 (control, n = 29). Other does were injected with 25IU equine chorionic gonadotrophin (eCG), followed by 0.2-ml gonadotrophin-releasing hormone (GnRH, n = 29) or 75IU human chorionic gonadotrophin (hCG, n = 29) per doe 48h later. After 60h of day 0, does in all groups were artificially inseminated (AI). In the 2nd experiment, does (n = 140) were mated (AI) after synchronization of estrus/ovulation with 25IU eCG, and 75IU hCG 48h later. On day 5 post-AI, does were injected with saline (control), 75IU hCG, 0.2ml GnRH, or 25IU eCG per doe. Injection of eCG with GnRH or hCG pre-AI significantly increased corpora lutea number, ovulation rate, total number/doe and recovery rate of embryos, viable embryos, hatched blastocysts, in vivo reproductive parameters, and concentration of progesterone and progesterone/estradiol 17-β ratio. Injection of eCG on day 5 post-AI significantly improved large and total follicle number, and in vivo reproductive efficiency. The corpora lutea number and impantation sites were significantly increased in the hCG and eCG groups. Fetal loss rate significantly increased only in the GnRH group. Under high ambient temperature, administration of eCG with hCG or GnRH injection pre-AI could be synchronized estrus/ovulation for improving in vivo and in vitro embryo production. In addition, pregnancy outcomes could be enhanced in rabbit does induced to ovulation by a single eCG or hCG dose on day 5 post-AI.

Effect of ovarian stimulation on the expression of piRNA pathway proteins.

PIWI-interacting RNAs (piRNAs) play an important role in gametogenesis, fertility and embryonic development. The current study investigated the effect of different doses of pregnant mare serum gonadotrophin/human chorionic gonadotrophin (PMSG/hCG) and repeated ovarian stimulation (OS) on the expression of the Mili, Miwi, Mael, Tdrd1, Tdrd9, qnd Mitopld genes, which have crucial roles in the biogenesis and function of piRNAs. Here, we found that after treatment with 7.5 I.U. PMSG/hCG and two repeated rounds of OS, both the mRNA and protein levels of Tdrd9, Tdrd1 and Mael showed the greatest decrease in the ovarian tissue, but the plasma E2 levels showed the strongest increases (p<0.05). However, we found that the Mitopld, Miwi and Mili gene levels were decreased significantly after treatment with 12.5 I.U. PMSG/hCG. Our results suggested that exogenous gonadotropin administration leads to a significant decrease in the expression of the Mili, Miwi, Mael, Tdrd1, Tdrd9 and Mitopld genes, which are critically important in the piRNA pathway, and the changes in the expression levels of Tdrd9, Tdrd1 and Mael may be associated with plasma E2 levels. New comprehensive studies are needed to reduce the potential effects of OS on the piRNA pathway, which silences transposable elements and maintains genome integrity, and to contribute to the safety of OS.

No effect of ovarian stimulation and oocyte yield on euploidy and live birth rates: an analysis of 12 298 trophectoderm biopsies

Does ovarian stimulation affect embryo euploidy rates or live birth rates (LBRs) after transfer of euploid embryos? Euploidy rates and LBRs after transfer of euploid embryos are not significantly influenced by gonadotropin dosage, duration of ovarian stimulation, estradiol level, follicle size at ovulation trigger or number of oocytes retrieved, regardless of a woman's age. Aneuploidy rates increase steadily with age, reaching >80% in women >42years old. The goal of ovarian stimulation is to overcome this high aneuploidy rate through the recruitment of several follicles, which increases the likelihood of obtaining a euploid embryo that results in a healthy conceptus. However, several studies have suggested that a high response to stimulation might be embryotoxic and/or increase aneuploidy rates by enhancing abnormal segregation of chromosomes during meiosis. Furthermore, a recent study demonstrated a remarkable difference in euploidy rates, ranging from 39.5 to 82.5%, among young oocyte donors in 42 fertility centres, potentially suggesting an iatrogenic etiology resulting from different stimulation methods. This is a retrospective cohort study that included 2230 in vitro fertilisation (IVF) with preimplantation genetic testing for aneuploidy (PGT-A) cycles and 930 frozen-thawed single euploid embryo transfer (FET) cycles, performed in our centre between 2013 and 2017. A total of 12 298 embryos were analysed for ploidy status. Women were divided into five age groups (<35, 35-37, 38-40, 41-42 and >42years old). Outcomes were compared between different durations of stimulation (<10, 10-12 and ≥13days), total gonadotropin dosages (<4000, 4000-6000 and >6000IU), numbers of oocytes retrieved (<10, 10-19 and ≥20 oocytes), peak estradiol levels (<2000, 2000-3000 and >3000pg/mL), and sizes of the largest follicle on the day of trigger (<20 and ≥20mm). Within the same age group, both euploidy rates and LBRs were comparable between cycles regardless of their differences in total gonadotropin dosage, duration of stimulation, number of oocytes harvested, size of the largest follicles or peak estradiol levels. In the youngest group, (<35years, n = 3469 embryos), euploidy rates were comparable between cycles with various total gonadotropin dosages (55.6% for <4000IU, 52.9% for 4000-6000IU and 62.3% for >6000IU; P = 0.3), durations of stimulation (54.4% for <10days, 55.2% for 10-12days and 60.9% for >12days; P = 0.2), number of oocytes harvested (59.4% for <10 oocytes, 55.2% for 10-19 oocytes and 53.4% for ≥20 oocytes; P = 0.2), peak estradiol levels (55.7% for E2 < 2000pg/mL, 55.4% for E2 2000-3000pg/mL and 54.8% for E2 > 3000pg/mL; P = 0.9) and sizes of the largest follicle (55.6% for follicles <20mm and 55.1% for follicles ≥20mm; P = 0.8). Similarly, in the oldest group (>42years, n = 1157 embryos), euploidy rates ranged from 8.7% for gonadotropins <4000IU to 5.1% for gonadotropins >6000IU (P = 0.3), from 10.8% for <10days of stimulation to 8.5% for >12days of stimulation (P = 0.3), from 7.3% for <10 oocytes to 7.4% for ≥20 oocytes (P = 0.4), from 8.8% for E2 < 2000pg/mL to 7.5% for E2 > 3000pg/mL (P = 0.8) and from 8.2% for the largest follicle <20mm to 8.9% for ≥20mm (P = 0.7). LBRs after single FET were also comparable between these groups. Although this large study (2230 IVF/PGT-A cycles, 12 298 embryos and 930 single FET cycles) demonstrates the safety of ovarian stimulation in terms of aneuploidy and implantation potential of euploid embryos, a multi-centre study may help to prove the generalisability of our single-centre data. These findings reassure providers and patients that gonadotropin dosage, duration of ovarian stimulation, estradiol level, follicle size at ovulation trigger and number of oocytes retrieved, within certain ranges, do not appear to significantly influence euploidy rates or LBRs, regardless of the woman's age. No external funding was received and there are no competing interests to declare. N/A.

In vitro and in vivo development of domestic cat embryos generated by in vitro fertilization after eCG priming and oocyte in vitro maturation

The objective of this study was to evaluate, in the domestic cat, the effect of ovarian stimulation with eCG prior to oocyte in vitro maturation (priming) on in vitro and in vivo development after in vitro fertilization (IVF). For this purpose, oocyte donors were either 1) treated with a single dose of 200 IU eCG four days before oocyte recovery (eCG group), or, 2) given no treatment before oocyte recovery (control group).Ovaries of both groups were collected by ovariohysterectomy (OVH) and cumulus-oocyte complexes (COCs) were recovered by slicing. Immature COCs from both groups were matured in vitro (IVM) for 26–28 h. IVF was done with refrigerated epididymal sperm. After 24 h co-incubation, presumptive zygotes were cultured in vitro for eight days. The rates of cleavage, morulae, blastocyst development and hatching were estimated. Some blastocysts were stained for total cell counting and others were used for gene expression analysis of pluripotency (OCT4, SOX2 and NANOG) and differentiation markers (CDX2 and GATA6). Additionally, to evaluate in vivo development, embryos from the eCG group were transferred at Day 5 and Days 7 or 8 of IVC to synchronized cat recipients. The results showed that, eCG priming increased significantly the rate of blastocyst development as compared to the control group (37.9 and 25.6%, respectively) (P < 0.05). No differences were observed in total cell number of blastocysts and hatching blastocysts (mean ± SD) between the eCG and control groups (420.6 ± 193.6 and 347.0 ± 237.1, respectively) (P > 0.05). In the gene expression analysis, blastocysts generated in the eCG group had higher expression of OCT4 than blastocysts from the control group (P < 0.05). However, no significant differences were observed in the relative expression of SOX2, NANOG, CDX2 and GATA6 (P > 0.05). Additionally, six embryo transfer (ET) procedures were done, three with Day 5 embryos and three with Day 7 or 8 embryos. Recipients from both ET groups delivered live kittens. The total pregnancy rate was 4/6 (67%), meanwhile the live birth rate was 2/6 (33%). In conclusion, eCG priming improved the rate of blastocyst development in vitro and increased relative expression of OCT4. These results demonstrate that eCG priming of oocytes donors before IVM improves oocyte competence, enhance in vitro embryo development and allows live births of healthy offspring after ET.

Predictive factors for biochemical pregnancy in intracytoplasmic sperm injection cycles

The aim of this study was to investigate which factors contribute to the incidence of biochemical pregnancy (BP) in intracytoplasmic sperm injection (ICSI) cycles. This cohort study included cycles performed from June 2010 to September 2016 in a private, university-affiliated IVF centre. Cycles were split into four groups, depending on the pregnancy outcomes: Clinical Pregnancy (CP, n = 903), Biochemical Pregnancy (BP, n = 55), Miscarriage (MI, n = 142) and Negative Pregnancy (NP, n = 2034). The effects of ovarian stimulation, laboratory data and seminal parameters on pregnancy outcomes were evaluated using adjusted general linear models. Discriminant analyses were conducted to construct a model for pregnancy prediction and to establish cut-offs for BP. The total sperm count (p = 0.035), total and progressive sperm motility (p = 0.001 and p = 0.023, respectively), total motile sperm count (TMSC, p = 0.029) and the endometrial thickness (p < 0.001) were lower among BP group cycles. Lower rates of high-quality cleavage-stage embryos were observed in the BP group compared to CP and MI groups (p < 0.001). In discriminant analyses, cut-offs for BP prediction were established for the following factors: endometrial thickness < 11 mm, sperm motility < 55.5% and total dose of follicle-stimulating hormone (FSH)> 2400 IU. The incidence of biochemical pregnancy was four times higher when the aforementioned factors did not meet the defined cut-offs. The combination of suboptimal endometrial development and poor seminal and embryo quality contribute to an increased incidence of biochemical pregnancy in ICSI cycles.

Controled Ovarian Stimulation with Urinary Gonadotrophins and Recombinant Gonadotrophins in Current Practice

The main objective of the this study is to compare the effectiveness of ovarian stimulation with recombinant gonadotrophins (FSHr) versus urinary gonadotrophins (hMG) within the number of oocytes obtained and the number of embryos in the two patient groups, according to age groups and associated pathologies. The study design was retrospective, monocentric. All patients (71) who have addressed the In Vitro Fertilization Clinic of the Prof. Dr. Panait Sirbu Clinical Obstetrics and Gynecology Hospital during 01.01.2010 and 31.12.2010 were included in this research. The total number of oocytes (8.5�3.9 vs 5.7�3.9, p=0.045), the number of fertilized oocytes (7.5�3.6 versus 3.6�2.8 p=0.004) and the number of embryos (6.6�3.5 versus 3.7�2.6, p=0.013) were higher in women under 35 years of age treated with recombinant FSHr compared to women treated with urinary gonadotrophins hMG. Ovarian stimulation with recombinant gonadotrophins provides a greater number of oocytes and a greater number of embryos in all patients, regardless of age and associated pathology. There are many variables that can influence the success rate, but the first variable that can be controlled is the choice of FSHr in daily clinical practice.

Analysis of Protein Oxidative Modifications in Follicular Fluid from Fertile Women: Natural Versus Stimulated Cycles.

Oxidative stress is associated with obstetric complications during ovarian hyperstimulation in women undergoing in vitro fertilization. The follicular fluid contains high levels of proteins, which are the main targets of free radicals. The aim of this work was to determine specific biomarkers of non-enzymatic oxidative modifications of proteins from follicular fluid in vivo, and the effect of ovarian stimulation with gonadotropins on these biomarkers. For this purpose, 27 fertile women underwent both a natural and a stimulated cycle. The biomarkers, glutamic semialdehyde (GSA), aminoadipic semialdehyde (AASA), Nε-(carboxymethyl)lysine (CML), and Nε-(carboxyethyl)lysine (CEL), were measured by gas-liquid chromatography coupled to mass spectrometry. Results showed that follicular fluid contained products of protein modifications by direct metal-catalyzed oxidation (GSA and AASA), glycoxidation (CML and CEL), and lipoxidation (CML). GSA was the most abundant biomarker (91.5%). The levels of CML amounted to 6% of the total lesions and were higher than AASA (1.3%) and CEL (1.2%). In the natural cycle, CEL was significantly lower (p < 0.05) than in the stimulated cycle, suggesting that natural cycles are more protected against protein glycoxidation. These findings are the basis for further research to elucidate the possible relevance of this follicular biomarker of advanced glycation end product in fertility programs.

P26.03: Perinatal outcomes in singleton pregnancies conceived by natural cycle as compared with conventional IVF and spontaneously conceived pregnancies

Worse perinatal outcomes associated to in vitro fertilisation (IVF) could be related to infertility per se, laboratory procedures and/or effects of ovarian stimulation. Natural cycle (NC) IVF permits to investigate the effect of IVF avoiding ovarian stimulation. Our objective was to assess the impact NC IVF on perinatal outcomes. Retrospective cohort study of singleton live births born from 2006 to 2016 in pregnancies obtained by NC IVF (n=71), conventional IVF (n=659), SC pregnancies in infertile (SC-I, n=194) and fertile (SC-F, n=136) couples. NC and conventional IVF pregnancies were obtained after a fresh transfer of cleavage stage embryos. Statistical comparisons were adjusted by maternal age. While IVF presented lower birthweight, NC showed similar birthweight (SC-F 3398g (394) vs SC-I 3260g (513) vs NC 3278g (493) vs IVF 3125g (517)*, p<0.001), birthweight centile (SC-F 50% (27) vs SC-I 55% (32) vs NC 51% (33) vs IVF 43% (31), p=0.004) and prevalence of low birthweight (SC-F 0.7% vs SC-I 7.2% vs NC 7% vs IVF 10.3%, p=0.004) as compared to SC. All infertile groups were associated with lower gestational age at delivery (SC-F 40.0 weeks (1.2) vs SC-I 39.0 weeks (3.1)* vs NC 39.3 weeks (1.4)* vs IVF 39.0 weeks (1.9)*, p<0.001) and higher prevalence of prematurity (SC-F 0.7% vs SC-I 5.7% vs NC 4.2% vs IVF 7%, p=0.04). This study shows preserved fetal growth in NC compared to conventional IVF. Future studies are warranted to better define the potential beneficial effect on perinatal outcomes associated to NC IVF.

Ovarian stimulation perturbs methylation status of placental imprinting genes and reduces blood pressure in the second generation offspring

Objective(s)Assisted reproductive technology (ART) is associated with DNA methylation dysfunction of offspring. However, it is unclear whether ovarian stimulation (OS) is responsible for DNA methylation dysfunction of offspring Study designWe built the first-generation (F1) and second-generation (F2) offspring mice model of ovarian stimulation. Bodyweight of F1 and F2 were measured. Expression levels of several imprinted genes (Impact, H19, Igf2, Plagl1, Mest, and Snrpn) in F1 placenta were tested. Methylation status of Plagl1 and H19 promoters was examined with bisulfite sequencing. Glucose tolerance, blood pressure, and heart rate were evaluated in F2 mice. ResultsThe OS F1 showed elevated bodyweights in the 2nd, 3rd and 4th weeks, but the difference disappeared in the 5th week. Plagl1 was down-regulated in OS F1. Promoters of Plagl1 and H19 were also hypermethylated in OS F1. F2 of OS mice had the similar bodyweight and glucose tolerance compared with the control F2. However, F2 of OS ♂F1+OS♀ F1 showed the decreased systolic pressure, diastolic pressure, and heart rate. ConclusionsOvarian stimulation perturbs expression levels and methylation status of imprinted genes in offspring. The effect of ovarian stimulation may be passed to F2.

Effectiveness of ovarian stimulation in women with reduced ovarian reserve

Effectiveness of ovarian stimulation in women with reduced ovarian reserve

103 OVARIAN STIMULATION OF ANESTROUS CATS WITH FOLLICLE-STIMULATING HORMONE IMPROVES OOCYTE QUALITY AND DEVELOPMENTAL CAPABILITY AFTER PARTHENOGENETIC ACTIVATION

The domestic cat is a valuable model for developing assisted reproductive technologies in endangered felids. However, the in vitro production of cat embryos is negatively affected during the anestrous season. It seems that a low FSH level or the few FSH receptors in the granulosa cells might affect oocyte quality. The objective of this research was to evaluate the effect of ovarian stimulation of anestrous cats with FSH on oocyte quality and developmental capability after parthenogenetic activation. For this, two experimental groups were made. The first (FSH group) consisted of 9 anestrous cats treated with a subcutaneous dose of 5 mg of porcine FSH, every 24 h, for 4 days. The second (control group) consisted of 10 untreated anestrous cats. The ovaries were recovered by ovariohysterectomy. Cumulus-oocyte complexes (COCs) were collected by slicing of the ovaries and classified morphologically as grade I (excellent), grade II (good), grade III (fair), and grade IV (poor) quality. Grade I and II COCs were matured in vitro during 30 h and matured oocytes were used for parthenogenetic activation. Matured oocytes were activated using 7% ethanol for 5 min, followed by incubation in 10 μg mL–1 cycloheximide and 5 μg mL–1 cytochalasin B for 5 h. Presumed zygotes were in vitro cultured in SOF for 8 days, and blastocyst and hatching blastocyst rates were assessed. The relative expression of pluripotency (OCT4, SOX2, and NANOG) and differentiation markers (CDX2 and GATA6) were evaluated by RT-qPCR in the blastocysts. The results were normalized using the geometric mean of GAPDH and SDHA. Statistical analysis was performed using the Wilcoxon non-parametric test. A total (mean ± s.d.) of 741 COCs (82.33 ± 36.61 per cat) were recovered from the FSH group and 660 COCs (66 ± 20.67 per cat) from the control group (P &gt; 0.05). The FSH group had a higher rate of grade I COCs and a lower rate of grade III and IV COCs (22.28% and 50.62%, respectively) than the control group (13.2% and 60.08%, respectively; P &lt; 0.05). However, no significant differences were found in grade II COCs between FSH (26.21%) and control groups (27.01%; P &gt; 0.05). Thereafter, 190 matured oocytes were activated in the FSH group and 198 in the control group. The FSH group had higher blastocyst and hatching blastocyst rates (30.52% and 13.15%, respectively) than the control group (13.13% and 1.01% respectively; P &lt; 0.05). The blastocysts from the FSH group had a higher cell number (± s.d.) than their counterparts from the control group (199.9 ± 53.6 and 121 ± 34.91, respectively; P &lt; 0.05). Furthermore, the blastocysts from FSH group had an increased relative expression of OCT4 and GATA6 (P &lt; 0.05). In conclusion, ovarian stimulation of anestrous cats with FSH improves oocyte quality and embryo developmental capability, which agrees with an increase in relative expression of OCT4 and GATA6.

Maternal age and ovarian stimulation independently affect oocyte mtDNA copy number and cumulus cell gene expression in bovine clones.

Does maternal ageing and ovarian stimulation alter mitochondrial DNA (mtDNA) copy number and gene expression of oocytes and cumulus cells from a novel bovine model for human IVF? Oocytes collected from females with identical nuclear genetics show decreased mtDNA copy number and increased expression of an endoplasmic reticulum (ER) stress gene with repect to ovarian stimulation, whilst differences in the expression of genes involved in mitochondrial function, antioxidant protection and apoptosis were evident in relation to maternal ageing and the degree of ovarian stimulation in cumulus cells. Oocyte quality declines with advancing maternal age; however, the underlying mechanism, as well as the effects of ovarian stimulation are poorly understood. Human studies investigating these effects are often limited by differences in age and ovarian stimulation regimens within a patient cohort, as well as genetic and environmental variability. A novel bovine cross-sectional maternal age model for human IVF was undertaken. Follicles were aspirated from young (3 years of age; n = 7 females) and old (10 years of age; n = 5 females) Holstein Freisian clones following multiple unstimulated, mild and standard ovarian stimulation cycles. These bovine cloned females were generated by the process of somatic cell nuclear transfer (SCNT) from the same founder and represent a homogeneous population with reduced genetic and environmental variability. Maternal age and ovarian stimulation effects were investigated in relation to mtDNA copy number, and the expression of 19 genes involved in mitochondrial function, antioxidant protection, oocyte-cumulus cell signalling and follicle development in both oocytes and cumulus cells. Young (3 years of age; n = 7 females) and old (10 years of age; n = 5 females) Holstein Freisian bovine clones were maintained as one herd. Stimulation cycles were based on the long GnRH agonist down-regulation regimen used in human fertility clinics. Follicle growth rates, numbers and diameters were monitored by ultrasonography and aspirated when the lead follicles were >14 mm in diameter. Follicle characteristics were analysed using a mixed model procedure. Quantitative PCR (qPCR) was used to determine mtDNA copy number and reverse transcriptase-qPCR (RT-qPCR) was used to measure gene expression in oocytes and cumulus cells. Method of ovarian stimulation (P = 0.04), but not maternal age (P > 0.1), was associated with a lower mtDNA copy number in oocytes. Neither factor affected mtDNA copy number in cumulus cells. In oocytes, maternal age had no effect on gene expression; however, ovarian stimulation in older females increased the expression of GRP78 (P = 0.02), a gene involved in ER stress. In cumulus cells, increasing maternal age was associated with the higher expression of genes involved in mitochondrial maintenance (TXN2 P = 0.008 and TFAM P = 0.03), whereas ovarian stimulation decreased the expression of genes involved in mitochondrial oxidative stress and apoptosis (TXN2 P = 0.002, PRDX3 P = 0.03 and BAX P = 0.03). The low number of oocyte and cumulus cell samples collected from the unstimulated cycles limited the analysis. Fertilization and developmental potential of the oocytes was not assessed because these were used for mtDNA and gene expression quantification. Delineation of the independent effects of maternal age and ovarian stimulation regimen on mtDNA copy number gene expression in oocytes and cumulus cells was enabled by the removal of genetic and environmental variability in this bovine model for human IVF. Therefore, these extend upon previous knowledge and findings provide relevant insights that are applicable for improving human ovarian stimulation regimens. Funding was provided by Fertility Associates and the University of Auckland. J.C.P. is a shareholder of Fertility Associates and M.P.G. received a fellowship from Fertility Associates. The other authors of this manuscript declare no conflict of interest that could be perceived as prejudicing the impartiality of the reported research.

Effect of ovarian stimulation with recombinant LH/ FSH, recombinant FSH and human menopausal gonadotrophin (hMG) in reducing apoptosis rate in cumulus cells of patients undergoing ICSI. a pilot study

The aim of the study is to investigate the effects of three different ovarian stimulation protocols, evaluating also the effects of recombinant LH and extractive hCG (with LH like activity) on the biological and clinical outcome. The apoptosis rate of the cumulus cell was used as molecular biomarker to assess the oocyte competence.

The effect of ovarian stimulation on the outcome of intrauterine insemination

Although intrauterine insemination is one of the oldest techniques in reproductive medicine, its significance is still controversially discussed. Many factors have been reported as influencing pregnancy rates after IUI. The aim of this retrospective analysis is to evaluate the success rate of repeated inseminations depending on the type of ovarian stimulation. Patients who underwent intrauterine insemination in Wiesbaden Kinderwunschzentrum between 1998 and 2010, not older than 45 years of age, with male subfertility were included in this study. On the whole, 5,346 inseminations on 2,180 patients were analyzed retrospectively. Females' mean age was 34.1, ranging from 19-45 years. In 433 cycles an insemination was performed during a natural cycle. 4,020 cycles were stimulated with recombinant FSH, 596 cycles with clomiphene, 194 with urinary FSH, 103 with HMG. The pregnancy rates range from 7.4% in the clomiphene group to 14.4% in the urinary FSH group. Clomiphene stimulation seems to offer the significantly lowest pregnancy rate (p = 0.03). The other types of stimulation do not differ significantly from each other concerning the pregnancy rate. Patients under 39 years of age do not profit from any ovarian stimulation. In 40 and more years of old patients, pregnancy rates are higher, if any stimulation was performed. To sum up, clomiphene stimulation showed to offer significantly lower pregnancy rates in comparison to the natural cycle, FSH stimulation and HMG stimulation in IUI treatment. While women younger than 40 seem not to profit from any ovarian stimulation, women over 40 do profit.