IU FSH Research Articles

Donor side effects experienced under minimal controlled ovarian stimulation with in vitro maturation vs. conventional controlled ovarian stimulation for in vitro fertilization treatment

To evaluate oocyte retrieval experiences and side effects under minimally controlled ovarian stimulation (COS) treatment for invitro maturation (IVM) of oocytes compared with conventional COS treatment. A retrospective survey study. Clinical invitro fertilization treatment center. Data were collected from subjects undergoing minimal COS treatment (n = 110; 600-800 IU follicle-stimulating hormone) for IVM of oocytes and conventional COS treatment for egg donation (n = 48; 1,800-2,600 IU follicle-stimulating hormone) from April 2022 to November2023. Minimal and conventional COS treatments. The most common side effects experienced during ovarian stimulation and after oocyte pick-up, satisfaction level, and the likelihood of recommending or repeating minimal or conventional COS. Statistical analysis included Mann-Whitney U test and χ2 tests, with a significance level. During minimal COS treatment, most subjects did not experience breast swelling (86%), pelvic or abdominal pain (76%), nausea or vomiting (96%), and bleeding (96%). After oocyte pick-up, the majority (75%) reported no pelvic or abdominal pain. The most common side effect was abdominal swelling (52%). Compared with conventional COS cycles, minimal COS subjects reported significantly less postretrieval pain, with 33% experiencing no pain (vs. 6%) and with a reduced severe level of pain (5% vs. 19%), leading to fewer subjects requiring pain medication (25% vs. 54%). Additionally, 85% of women were very satisfied with minimal stimulation treatment and would recommend or repeat the treatment. Reducing the hormonal dose for ovarian stimulation has a beneficial effect on subjects, suggesting the combination of minimal COS treatment with IVM techniques is a well-tolerated alternative for women who cannot or do not wish to undergo conventionally controlled ovarian hyperstimulation treatment.

An Innovative Approach of HCG Trigger with Concurrent 1500 IU HCG and 450 IU FSH Utilization for OHSS Avoidance in High Risk Patients - Is it Superior to Dual Trigger

An Innovative Approach of HCG Trigger with Concurrent 1500 IU HCG and 450 IU FSH Utilization for OHSS Avoidance in High Risk Patients - Is it Superior to Dual Trigger

Role of aquaporin 3 in reproductive performance of dairy goats after repeated estrus synchronization stimulation.

This study explored the specific molecular mechanisms through which repeated estrus synchronization (ES) treatments reduce the reproductive performance of dairy goats. Ninety-six goats (n=24/group) were randomly assigned to two groups receiving ES treatments thrice every fortnight (3- equine chorionic gonadotropin [eCG] and 3-follicle stimulating hormone [FSH] groups) and two groups receiving one ES treatment (1-eCG and 1-FSH groups). ES treatments of 1- and 3-eCG goats were performed via the intravaginal insertion of a controlled internal drug release (CIDR) device containing 300 mg progesterone (P4), followed by 300 IU eCG injections 48 h before CIDR withdrawal. The 1- and 3-FSH goats received CIDR for 10 d, followed by 50 IU FSH and 100 μg PGF2α within 12 h of CIDR withdrawal. Ovaries of three goats in estrus from both groups were harvested for analysis. Subsequently, all the goats in estrus were artificially inseminated twice. Consequently, 3-eCG and 3-FSH goats showed a considerably reduced estrus rate and litter size than 1-eCG and 1-FSH goats. AQP3 mRNA and protein expression were significantly higher in the 3-eCG and 3-FSH groups than in the 1-eCG and 1-FSH groups. AQP3 overexpression led to cell apoptosis and decreased steroid hormone secretion ability of ovarian granulosa cells. Moreover, it resulted in a decrease in maturation and cleavage rates after parthenogenetic activation and in vitro fertilization, respectively. AQP3 gene was involved in reducing the reproductive performance of repeated ES-treated dairy goats. These findings provide a theoretical foundation for the effective use of reproductive hormones in breeding techniques for livestock.

Superovulation of high-producing Holstein lactating dairy cows with human recombinant FSH and hMG

Superovulation of high-producing dairy cows is a challenging subject in dairy farms with respect to the cost, dose and type of gonadotropin. The objectives of this study were to compare three gonadotropin products: Folltropin-V® (highly purified FSH with porcine origin), Cinnal-f® (recombinant human FSH) and Menotropins® (hMG) for superovulation in high-producing Holstein lactating dairy cows and to investigate the pregnancy outcomes achieved following transferring embryos recovered from donors treated with different gonadotropins. Healthy high-producing Holstein lactating dairy cows (n = 30; milk production: 46.35 ± 8.78 kg; parity: 2–4; days in milk: 80–130 days) without any puerperal problems were selected as donors. On Day 10 after estrus (Day 0 of superovulation), donors (10 cows in each experimental groups) received Folltropin-V® (400 mg NIH, dissolved in 20 ml), Cinnal-f® (20 vials; each vial of 1 ml contains 75 IU Follitropin alfa) and Menotropins ® (20 ampules; each ampule of 1 ml contains 75 IU FSH and 75 IU LH), administered twice daily, in decreasing doses (4,4; 3,3; 2,2; 1,1 ml), over 4 days. On Day 2 of superovulation, donors received 3 doses of prostaglandin F2α analogue, 6 h apart. They were inseminated twice with a frozen semen at 12 and 24 h after standing estrus. Concurrent with the second insemination, donors received 2500 IU hCG (Karma Pharmatech GmbH, Germany). On Day 7 after standing estrus, superovulatory responses (number of CLs, total ova/embryos and transferable embryos) were recorded and Code 1 embryos, recovered from each treated donors, were transferred to synchronized heifers. Pregnancy was detected on Day 30 and 60 after AI. Gestation length, the number and weight of live births were recorded. Data were analyzed using Proc GLM, Proc Mixed and Proc Genmod of SAS. The respective number of corpora lutea, total number of ova/embryos and transferable embryos were not different among donors received Cinnal-f (25.5 ± 3.01, 11.2 ± 2.77, 5.1 ± 0.86), Menotropins (24.0 ± 3.21, 9.0 ± 2.04, 6.3 ± 1.74) and Folltropin-V (20.3 ± 3.21, 8.9 ± 1.90, 5.1 ± 1.16; P > 0.05). Pregnancy rates on Day 30 was similar among treatment groups (P > 0.05). However, pregnancy rates on Day 60 and the number of calves born healthy was less in heifers that received embryos from Cinnal-f treated donors (P < 0.05). In conclusion, Cinnal-f and Menotropins could provide similar superovulatory response to Folltropin-V for superovulation of high-producing Holstein lactating dairy cows.

P–273 Effects of ovulation induction with GnRH Agonist (GnRHa) on oocyte and embryo quality at the mitochondrial level: A retrospective and experimental study

Abstract Study question Does the GnRHa trigger improve oocyte and embryo quality in patients younger than 40, and do mtUPPR have a role? Summary answer GnRHa trigger improves oocyte nuclear/cytoplasmic maturation, blastocyst utilization and downregulates HSP60 levels and upregulates ATF5 levels compared to hCG trigger. GnRHa trigger suppresses mitochondrial stress. What is known already hCG has been used for decades to achieve final oocyte maturation and, thereby, correct oocyte retrieval timing in connection with ovarian hyperstimulation protocols. As an alternative to hCG, a GnRH agonist has been used to trigger the endogenous release of LH (and FSH) in a fashion resembling the mid-cycle surge of gonadotrophins. GnRHa is as effective as hCG for the induction of ovulation. It has been very well known that the GnRHa trigger improves oocyte nuclear maturation, embryo quality, and implantation rate, but the underlying mechanism remains unknown. Study design, size, duration 3054 women younger than 40; oocytes retrieved more than 10 (up to 20) analyzed. Male infertility was excluded. Ovulation triggered either by hCG (n = 1368) or GnRHa (1668). Female mice were divided into three groups as control, hCG-treated and GnRHa-treated group. Superovulation was performed by FSH + hCG or GnRHa. Oocytes were collected 13 hours after hCG/GnRHa injection. ATF5, BiP, and HSP60 levels were analyzed by Western blot. Statistical analysis was performed using Student’s t-test. Participants/materials, setting, methods This study has two parts. i) RCT and ii) Experimental. In the experimental part, three months old female BALB/C mice (25–30 g) were used and divided into three groups (n = 20/group) as control, hCG-treated and GnRHa-treated group. Superovulation was performed by administering an injection of 5 IU FSH (i.p.) and hCG (i.p.) or GnRHa (20 mg/kg) i.m. Oocytes were collected 13 hours after hCG/GnRHa injection. ATF5, BiP, and HSP60 levels were analyzed by Western blot. Main results and the role of chance The mean age (34.8 vs. 35.2 years), total gonadotropin dose (2176 vs. 2230 IU), and the number of oocytes picked up (14.9 vs. 13.4) were not statistically different among GnRHa and hCG group, respectively. No LH rise or any OHSS was noticed in any groups. Oocyte maturation (79.8% vs. 75.9%), oocyte diameter (as a marker of cytoplasmic maturity) (10198 µm2 and 9474 µm2), fertilization rate (78% vs. 72%), and embryo utilization rate (52% vs. 47.2%) were significantly higher in GnRHa group compared to hCG group, respectively. HSP60 level (activated by mtUPR) was statistically higher in the hCG group compared to the GnRHa group (55% vs. 22%, p &amp;lt; 0.05 respectively). On the other hand, the ATF5 level was significantly higher in the GnRHa group than the hCG group (p &amp;lt; 0.0001). Limitations, reasons for caution The limitation is that this is a proof-of-concept study to reveal the mechanism of good embryo quality with GnRHa trigger. Wider implications of the findings: This application offers convenience and simplifies the IVF protocol with a better oocyte and embryo quality while reducing Ovarian Hyperstimulation Syndrome (OHSS) risk during IVF care Trial registration number Not applicable

Follitropin alpha versus beta in a first GnRH antagonist ICSI cycle: a retrospective cohort study

Research questionAre cumulative live birth rates (CLBR) after follitropin alpha (Ovaleap®) and follitropin beta (Puregon®) similar when used for ovarian stimulation with ICSI (intracytoplasmic sperm injection) in a first-rank gonadotrophin-releasing hormone (GnRH) antagonist protocol? DesignRetrospective single-centre cohort study including 832 infertile patients undergoing ovarian stimulation with a daily dose of 150–225 IU FSH in their first ICSI cycle at a tertiary referral centre between July 2016 and July 2019. Of those, 349 patients used Ovaleap and 483 patients received Puregon. ResultsBaseline characteristics were not statistically different between the groups. The duration of stimulation was slightly longer in the Ovaleap group (10.6 ± 1.7 versus 10.3 ± 1.6 days; P = 0.012). The number of mature oocytes was not statistically different and there was no significant difference in fertilization rate or embryo utilization rate between the two groups. After fresh embryo transfer, biochemical pregnancy rate (137/349 [39.3%] versus 186/483 [38.5%]) as well as clinical pregnancy rate (105/349 [30.1%] versus 152/483 [31.5%]) were comparable (P = 0.83 and 0.67, respectively). Live birth rate (LBR) after fresh embryo transfer (94/349 [26.9%] versus 141/483 [29.2%]; P = 0.48) and CLBR (199/349 [57.0%] versus 287/483 [59.4%]; P = 0.49) were not significantly different. Multivariable regression analysis revealed that the type of gonadotrophin was not associated with CLBR (P = 0.28). ConclusionThis retrospective study shows no significant difference in CLBR between Ovaleap and Puregon in patients undergoing their first GnRH antagonist ICSI cycle.

FSH dose is negatively correlated with number of oocytes retrieved: analysis of a data set with ~650,000 ART cycles that previously identified an inverse relationship between FSH dose and live birth rate.

To evaluate whether total FSH dose was negatively correlated with number of oocytes retrieved in a large data set where previously, a negative correlation between FSH dose and live birth rate was identified. Data from 650,637 fresh autologous in vitro fertilization (IVF) cycles reported to the Society for Assisted Reproductive Technology between 2004 and 2012 were included. Logistic regression analysis was performed to determine if the relationship between total FSH dose used during ART with number of oocytes retrieved was impacted by the patient's health prognosis, age, BMI, ovarian stimulation protocol, or infertility diagnosis. The number of oocytes retrieved was negatively correlated with FSH dose (P < 0.0001). Regardless of patient prognosis, age, BMI, ovarian stimulation protocol, and infertility diagnosis, the highest number of oocytes retrieved was in the 1001-2000 IU FSH group, and was 36-51% lower in the > 5000 IU compared with the optimal, 1001-2000 IU, FSH groups. Overall, ~80% of patients received FSH doses outside of the optimal FSH dose. Moreover, 61% of good prognosis patients (excludes individuals likely prescribed higher FSH doses) received doses exceeding the optimal dose range. The inverse relationship between FSH dose and the number of oocytes retrieved independent of patient age or health implies that excessive FSH doses during ART may be detrimental to oocyte retrieval.

Pregnancy rate of intrauterine insemination (IUI) - is it affected by inseminated motile sperm count and stimulation protocol?

Objective To compare IUI pregnancy rate by partner and donor sperm according to inseminated motile sperm count and ovulation induction protocol Design Retrospective study by a private IVF center Materials and methods 3,028 IUI cycles (2,284 cycles with partner sperm and 744 cycles with donor sperm) were analyzed. Ovulation induction was done using 5 mg of letrozole (Let) on cycle days 3 through 7, with 250 mcg recombinant hCG injected as instructed after follicle monitoring is done on day 12. A sequential (Seq) stimulation involves Let protocol, followed by 150 IU FSH daily from cycle day 7. Ultrasound monitoring is done on day 10 to determine further medication dosing, GnRH antagonist initiation, and monitoring. A controlled ovarian hyperstimulation (COH) cycle involves daily FSH injections at doses between 150 and 300 IU FSH, with monitoring beginning on day 7 to determine further medication dosing, GnRH antagonist initiation, and monitoring. All IUI procedures were performed approximately 36 hours after trigger. Pre-washed frozen sperm was thawed by agent's thawing protocol. Total motile sperm for insemination was counted after finishing sperm wash process. The statistical analysis was performed using t-test, chi-square, or z-test. A p value of Results IUI pregnancy rate by using partner sperm was significantly lower (n=106, 3.8%) when inseminated motile sperm is Conclusions IUI pregnancy rate is not affected by sperm count if at least 3 million motile sperm are inseminated. Pregnancy rate from natural cycle is low in both partner sperm and donor sperm cycle. There is no significant difference of pregnancy rate between ovulation induction protocols although the pregnancy rate in Let group is slightly lower than that in COH or Seq group.

Receptor Binding Inhibitor Suppresses Carcinogenesis of Cervical Cancer by Depressing Levels of FSHR and ERβ in Mice.

FSH Receptor Binding Inhibitor (FRBI) blocked the binding of FSH to FSHR. Our initial study revealed FRBI reduced the maturation rate, enhanced the apoptosis of sheep Cumulus-Oocyte Complex (COCs). Little is known about whether FRBI modulates ERβ and FSHR levels in the normal uterine and cancerous tissues. The present study aimed to evaluate the FRBI effects on the expressions of Estrogen Receptor-beta (ERβ) and FSH receptor (FSHR) in the uteri. 150 mice were assigned to FRBI+FSH (COM), FSH and control groups (CG). Mice of COM-1, COM-2 and COM-3 groups were simultaneously intramuscularly injected with 500, 750 and 1000 µg FRBI with 10 IU FSH, respectively for five days. Western blotting and qPCR were utilized to determine the expression of ERβ and FSHR. In comparison with FSH group, uterine lumen and glands of COM groups became narrow. The uterine wall and endometrial epithelium were thinned, and uterine lumen became narrow. Epithelial cells were decreased. Uterine wall thicknesses of COM-1, COM-2 and COM-3 groups were reduced by 6.49%, 14.89% and 15.69% on day 30 as compared with FSH group. Uterine perimetrium thicknesses of COM-1, COM-2 and COM-3 groups were reduced by 16.17%, 17.93% and 19.92% on day 20 in comparison with FSH group. Levels of FSHR mRNAs and proteins of COM-1, COM-2 and COM-3 groups were less than FSH group on days 20 and 30 (P<0.05). ERβ protein of COM-3 group was less than FSH group. Serum estradiol (E2) and FSH concentrations of COM-2 and COM-3 were lower than that of FSH group on day 30. FRBI could decrease UWT and UPT, also block the uterine development, decline expression levels of ERβ and FSHR protein. Additionally, FRBI reduced the secretion of secretion of FSH and E2. Downregulating expression of FSHR and ERβ may be a potential treatment regimen for cervical cancer patients.

Melatonin-induced demethylation of antioxidant genes increases antioxidant capacity through RORα in cumulus cells of prepubertal lambs

Physical damage and oxidative stress may occur in prepubertal cumulus cells, due to insufficient glutathione synthesis. To determine potential epigenetic mechanisms related to antioxidant effects of melatonin on ovine prepubertal cumulus cells, 30 lambs, 4-wk-old were randomly allocated into two groups: a control (C, n = 20) group and a melatonin (M, n = 10) group given a subcutaneous implant containing 18 mg melatonin. All lambs were superovulated (250 IU FSH and 250 IU eCG). Cumulus cells from germinal vesicle stage cumulus oocyte complexes (COCs) were collected by ovarian follicular aspiration and dissociated with hyaluronidase. Compared to the C group, the M group had greater superovulation, better antioxidant capacity, a higher proportion of fully expanded COCs and a lower proportion of apoptotic cumulus cells (P < 0.05). Melatonin up-regulated mRNA expression of genes for melatonin receptors MT1 and nuclear binding site RORα, antioxidants (SOD1, GPx4 and CAT) and cumulus cell expansion (PTX3, HAS2 and PTGS2), as well as Bcl2, but down-regulated expression of Bax (P < 0.05). Regarding epigenetics, there were less methylation at five CpG sites of SOD1, three CpG sites of GPx4 and two CpG sites of CAT in M versus C groups (P < 0.05), leading to lower total methylation of SOD1, GPx4 and CAT promoters region on M group (P < 0.05). In a mechanistic study, addition of MT1 or RORα antagonist increased ROS and MDA concentrations, but decreased T-AOC, GPx, CAT and T-SOD concentrations (P < 0.05), whereas there were no significant difference between the melatonin and MT2 antagonist treatment groups for T-AOC, GPx, CAT and T-SOD concentrations. Furthermore, addition of RORα agonist decreased total DNA methylation of SOD1, GPx4 and CAT, with no significant difference after MT1 agonist treatment. These studies provided new information regarding epigenetic mechanisms by which melatonin promoted ovine prepubertal cumulus cells antioxidant through RORα, both in vitro and in vivo.

Should corifollitropin alfa be offered to patients with “genuine” poor response to controlled ovarian hyperstimulation?

Objective: To examine whether poor ovarian response (POR) patients during conventional IVF/ intracytoplasmic sperm injection (ICSI) cycle, may benefit from multiple-dose GnRH-antagonist protocol with 150 mg of corifollitropin alfa via a cohort historical study. at a Tertiary, University affiliated Medical Center. Materials and Methods: Eighteen POR patients, defined according to the Bologna criteria, who underwent a subsequent 150 mg corifollitropin alfa cycle, within three months of the previous failed conventional IVF/ICSI cycle were included. The elimination of bias in this selection, for the purposes of this study, was achieved by including only a subgroup of “genuine” poor responder patients, those who yielded up to three oocytes following COH with a minimal gonadotropin daily dose of 300 IU. One hundred fifty mg corifollitropin alfa, administered on day 2-3 of the menstrual cycle, followed highly purified human menotropin (HP-hMG) or rFSH + rLH from stimulation day 5-6, within a flexible multiple-dose GnRH-antagonist COH cycle. Pregnancy rate, number of oocytes retrieved, number of embryos transferred, and COH variables were assessed. Results: The corifollitropin alfa COH protocol provided a non-significant one more oocyte, with no pregnancies. Considering the equivalence of 150 mg corifollitropin alfa to 2,100 IU of FSH, offering corifollitropin has no cost-effective advantages. Discussion: The corifollitropin alfa COH is of no benefit for “genuine” POR and alternative strategies, such as increasing the daily FSH dose or proceeding to egg-donation, should be seriously considered for this population.

FSH and eCG impact follicles development and expression of ovarian FSHR and caspase-9 in mice.

The study aimed to investigate the effects of FSH and eCG on the ovarian and follicular development, expression levels of FSHR and caspase-9 of ovaries in vivo. One hundred and five prepuberty mice were allocated into FSH-1, FSH-2, FSH-3, eCG-1, eCG-2, eCG-3 groups and control group (CG). Mice in FSH-1, FSH-2 and FSH-3 were intramuscularly injected with 5, 10 and 20 IU FSH twice (on day 0 and 4), respectively. Mice in eCG-1, eCG-2 and eCG-3 were intraperitoneally injected with 10, 20 and 40 IU eCG on day 0 and 4. Mice in the CG were injected with 0.5 ml normal saline on day 0 and 4. Left and right ovaries of each mouse were dissected aseptically on days 7, 14 and 21, respectively. The results showed that on days 14 and 21 the ovarian sizes and follicle numbers of FSH-3 and eCG-3 groups were greater than CG (P<0.05). FSHR mRNA of FSH-2 and eCG-1 were higher than CG on days 14 and 21 (P<0.05). FSHR proteins of FSH-3 were higher than CG on days 14 and 21 (P<0.05). Caspase-9 mRNA in FSH and eCG groups was less than CG. There were positive correlations between follicle numbers and FSH and eCG doses. FSHR protein expressions had positive correlations between ovarian weights and sizes of ovary and follicle numbers (r=0.971, P<0.05) in FSH-treated mice. Serum FSH concentrations of FSH-2, FSH-3, eCG-2 and eCG-3 groups were greater than that of CG. In conclusion, eCG and FSH promoted the ovarian development, follicle genesis, FSH secretion, FSHR mRNA and protein expressions in ovaries of mice. FSH and eCG inhibited the expression of ovarian caspase-9 mRNA.

A novel technique for in vitro maturation of sheep oocytes in a liquid marble microbioreactor.

The aim of this work was to develop a microbioreactor using liquid marble (LM) as a novel system for oocyte in vitro maturation (IVM) in small volumes. Cumulus-oocyte complexes (COCs) obtained from slaughterhouse sheep ovaries were in vitro matured in a LM system prepared by placing a drop (30μl containing 10 COCs) suspended in TCM 199 supplemented with 10% (v/v) oestrus sheep serum (OSS) and 0.1IU FSH and LH onto a polytetrafluoroethylene (PTFE) particle bed (LM group). As a control group (CTRL group), COCs were in vitro matured in standard volume and conditions (600μl of IVM medium in a four-well dish). After 24-h culture at 38.5°C in 5% CO2 in air, COCs were released from LM and the following parameters were evaluated: (a) percentage of MII oocytes, (b) oocyte developmental competence following in vitro fertilization (IVF) or parthenogenetic activation (PA) and embryo culture for 8days in synthetic oviductal fluid (SOF) medium at 38.5°C in 5% O2, 5% CO2, and 90% N2. The results indicated similar percentage of MII oocytes in LM and CTRL groups (88.0 vs. 92.0%). No differences were observed in blastocyst rate after IVF (LM 47.5% vs. CTRL 50.2%, P=0.637) or PA (LM 44.4% vs. CTRL 48.3%, P=0.426). The results indicate that LM microbioreactor is a viable technique that provides a suitable microenvironment to induce oocyte in vitro maturation.

Adjuvant sildenafil therapy in poor responders undergoing in vitro fertilization: A prospective, randomized, double-blind, placebo-controlled trial

Abstract Objective: To evaluate the effect of adjuvant sildenafil on ovarian responsiveness in low responders undergoing IVF. Patient(s) and methods: Prospective randomized, double-blind, placebo controlled study was conducted at Obstetrics and Gynecology department, Tanta university hospital and Center of Assisted Reproduction, Om El-kora Hospital, Tanta, Egypt. Sixty patients were classified as low responders undergoing IVF. Supplementation with sildenafil (50 mg daily) or placebo to a gonadotropin releasing hormone antagonist protocol was provided. Result(s): There were no significant differences in total number of 75 IU FSH ampoules used (60.025 ± 5.52) versus (66.025 ± 4.51), the number of mature follicles recruited (3.35 ± 1.137) versus (2.95 ± .826), and the number of oocytes retrieved (3.95 ± 1.395) versus (3.65 ± 1.137) or cycle cancelation rates (26.7% in the treatment group versus 23.3% in placebo group). Conclusion: Adjuvant sildenafil does not enhance ovarian responsiveness in cases of previous low ovarian response to controlled ovarian hyperstimulation.

Timing of human preimplantation embryonic development is confounded by embryo origin.

STUDY QUESTIONTo what extent do patient- and treatment-related factors explain the variation in morphokinetic parameters proposed as embryo viability markers?SUMMARY ANSWERUp to 31% of the observed variation in timing of embryo development can be explained by embryo origin, but no single factor elicits a systematic influence.WHAT IS KNOWN ALREADYSeveral studies report that culture conditions, patient characteristics and treatment influence timing of embryo development, which have promoted the perception that each clinic must develop individual models. Most of the studies have, however, treated embryos from one patient as independent observations, and only very few studies that evaluate the influence from patient- and treatment-related factors on timing of development or time-lapse parameters as predictors of viability have controlled for confounding, which implies a high risk of overestimating the statistical significance of potential correlations.STUDY DESIGN, SIZE, DURATIONInfertile patients were prospectively recruited to a cohort study at a hospital fertility clinic from February 2011 to May 2013. Patients aged <38 years without endometriosis were eligible if ≥8 oocytes were retrieved. Patients were included only once. All embryos were monitored for 6 days in a time-lapse incubator.PARTICIPANTS/MATERIALS, SETTING, METHODSA total of 1507 embryos from 243 patients were included. The influence of fertilization method, BMI, maternal age, FSH dose and number of previous cycles on timing of t2-t5, duration of the 2- and 3-cell stage, and development of a blastocoel (tEB) and full blastocoel (tFB) was tested in multivariate, multilevel linear regression analysis. Predictive parameters for live birth were tested in a logistic regression analysis for 223 single transferred blastocysts, where time-lapse parameters were investigated along with patient and embryo characteristics.MAIN RESULTS AND THE ROLE OF CHANCEModerate intra-class correlation coefficients (0.16–0.31) were observed for all parameters except duration of the 3-cell stage, which demonstrates that embryos from one patient elicit clustering at a patient level. No single patient- and treatment-related factor was found to systematically influence the timing from cleavage to blastocyst stage, which indicates that no individual patient-related factor can be identified that separately explains the clustering throughout the entire developmental stages. The blastocyst parameters were more affected by patient-related factors than cleavage stage parameters, as tEB occurred significantly later with older age (0.29 h/year (95% confidence interval: CI 0.03; 0.56)), while both tEB and tFB occurred significantly later with increasing dose of FSH (tEB: 0.12 h/100 IU FSH (95% CI 0.01;0.24); tFB 0.14 h/100 IU FSH (95% CI 0.03;0.27)) and with more previous attempts (tEB: 1.2 h/attempt (95% CI 0.01;2.5); tFB 1.4 h/attempt (0.10;2.7)). Fertilization method affected timing of the first division, with ICSI embryos cleaving significantly faster than IVF embryos (−3.6% (95% CI −6.4; −0.77)), whereas no difference was found in the subsequent divisions. The univariable regression analysis identified female age, cumulative FSH dose, degree of blastocyst expansion, score of the inner cell mass and timing of full blastocyst formation as predictors of live birth. The timing of full blastocyst formation (tFB) did not remain significant when adjusting for age, number of previous cycles and cumulative FSH dose, which were the parameters shown to influence tFB in the mixed regression model.LIMITATIONS, REASONS FOR CAUTIONOnly good prognosis patients were enrolled, so these results may not be generalized to all infertile women. Not all patient-related factors were investigated.WIDER IMPLICATIONS OF THE FINDINGSOur findings underline the importance of treating embryos as dependent observations and suggest a high risk of patient-based confounding in retrospective studies. The impact of confounders and the embryo origin needs to be addressed in order to apply appropriate statistical models in observational studies. Furthermore, this observation emphasizes the need for RCTs for evaluating use of time-lapse parameters for embryo selection.STUDY FUNDING/COMPETING INTERESTSFunding for the cohort study was provided by the Lippert Foundation, the Toyota Foundation, the Aase og Einar Danielsen foundation and NordicInfu Care research grant. Research at the Fertility Clinic, Aarhus University Hospital is supported by an unrestricted grant from MSD and Ferring. K.K. is funded by a grant from the Danish Council for Independent Research Medical Sciences. The authors declare no competing interest.

66 CUMULUS-OOCYTE-COMPLEX SECRETIONS FROM THE FIRST 8 HOURS OF IN VITRO MATURATION AFFECT OOCYTE DEVELOPMENTAL COMPETENCE

Mammalian oocytes are surrounded by cumulus cells, forming a structure known as the cumulus-oocyte complex (COC). Cumulus cells play important protective functions during oocyte maturation, for example, protecting the oocyte against reactive oxygen species. However, it is not yet fully understood how the cumulus complex modulates the developmental competence of the enclosed oocyte. It was investigated whether direct contact between an oocyte and its cumulus cells is essential throughout the maturation process. To this end, bovine oocytes aspirated from ovarian follicles were matured in vitro. Eight hours after the onset of maturation the cumulus cells were removed, and the oocytes either placed back in the original medium or cultured further in fresh maturation medium. In all experiments, COCs/oocytes were matured for 23 h in M199 supplemented with 0.05 IU FSH and penicillin/streptomycin. All experiments were performed in triplicate, with 35 to 45 COCs per group. Student’s t-test was used for a paired comparison. Denudation after 8 h and return to the same maturation medium had no effect on the cleavage rate (93%) compared with culture without denudation (90.7%). Only if the oocytes were transferred to fresh medium did the cleavage rate decrease slightly (75.4%; P = 0.038). By contrast, blastocyst formation was reduced nearly four times if COCs were denuded before being returned to the medium, compared with controls (14% v. 50.8%; P &lt; 0.001). If the oocytes were transferred to fresh medium after denudation, very few blastocysts resulted (0.9%; P &lt; 0.001). In a second study, oocytes denuded immediately after removal from the follicle were matured in the absence or presence of cumulus cells in a Corning® Transwell® system. Culturing denuded oocytes in the presence of cumulus cells resulted in similar cleavage rates (83.5%) to control conditions (84.8%). However, blastocyst formation was markedly lower (4.3%) than for controls (29.6%; P = 0.003). We conclude that COCs secrete substances during the first 8 h of maturation that are beneficial for oocyte acquisition of developmental competence. Moreover, intimate contact between the cumulus cells and oocyte is essential. This work was supported by EU FP7 EpiHealthNet, PITN-GA-2012–317146.

450 IU versus 600 IU gonadotropin for controlled ovarian stimulation in poor responders: a randomized controlled trial

To compare the outcomes of controlled ovarian stimulation/in vitro fertilization cycles using 450 IU and 600 IU gonadotropin per day in women at risk of poor ovarian response. Prospective randomized controlled nonblinded study. University-affiliated private IVF center. Women considered to be at risk of poor ovarian response: aged <41 years with basal FSH >10 IU/L, antimüllerian hormone <1 ng/mL, antral follicle count ≤ 8, or a previous IVF cycle with ≥ 300 IU/d gonadotropin that resulted in a cancellation, <8 follicles, or <5 oocytes. A total of 356 patients underwent a microdose GnRH agonist flare-up IVF/intracytoplasmic sperm injection protocol with a fixed daily dose of either 450 IU FSH (n = 176) or 600 IU FSH (n = 180) equally divided between Menopur and Bravelle. Number of mature oocytes retrieved. The two groups were similar in terms of age, ovarian reserve, cause of infertility, duration of stimulation, and cycle cancellation rate. There were no significant differences in the number of metaphase II oocytes retrieved (4 [range 0-6] vs. 4 [range 2-7]), fertilization rate (62.4% vs. 57.0%), biochemical pregnancy rate (20.5% vs. 22.9%), clinical pregnancy rate (16.4% vs. 18.3%), and implantation rate (29.8% vs. 30.4%) between the 450 IU and 600 IU groups, respectively. Gonadotropin of 600 IU/d does not improve outcome of IVF cycles compared with 450 IU/d in women at risk of poor ovarian response. NCT00971152.

Diminished ovarian reserve in the United States assisted reproductive technology population: diagnostic trends among 181,536 cycles from the Society for Assisted Reproductive Technology Clinic Outcomes Reporting System

To evaluate trends in diminished ovarian reserve (DOR) assignment in the Society for Assisted Reproductive Technology (SART) Clinic Outcomes Reporting System database and to evaluate its accuracy in predicting poor ovarian response (POR) as defined in European Society of Human Reproduction and Embryology's Bologna criteria (2011). Retrospective cohort study. Not applicable. A total of 181,536 fresh, autologous ART cycles reported to SART by U.S. clinics in 2004 and 2011 (earliest and most recent available reporting years). None. DOR assignment was the primary exposure. POR, defined as cycle cancellation for poor response or less than 4 oocytes retrieved after conventional gonadotropin stimulation (>149 IU FSH daily), was the primary outcome. Secondary outcomes were live birth and number of oocytes retrieved. DOR prevalence, power of DOR and FSH (</≥12 mIU/mL) to predict POR, and live birth in POR cycles were also calculated. DOR prevalence increased from 19% to 26% from 2004 to 2011. Among cycles clinically assigned as DOR, incidence of POR decreased from 32% to 30%, and live birth improved from 15% to 17%. Comparing basal FSH ≥12 versus clinical assignment of DOR, basal FSH had a higher specificity (92.2% vs. 81.6%) and positive predictive value (38.3% vs. 30.9%) for predicting POR. Live birth among POR cycles was 4%. DOR diagnosis is increasing, and accuracy remains poor, despite the availability of additional diagnostic parameters such as antral follicle count and antimüllerian hormone. POR entailed poor outcomes, but the majority of patients clinically assigned as DOR did not experience POR. Development and use of more accurate predictors of POR are needed to minimize patient distress resulting from overdiagnosis.

299 EFFECT OF ESTRADIOL-17β AND FOLLICLE STIMULATING HORMONE ON THE IN VITRO MATURATION OF PORCINE OOCYTES DERIVED FROM SMALL FOLLICLES

In porcine cumulus-oocyte complexes (COC) from middle follicles (MF: 3–6 mm in diameter), FSH is known to induce the resumption of meiosis and accompanied by transactivate of the EGF receptor and activation of MAPK3/1 in the cumulus cells. The aim of the current study was to examine the effect of oestradiol-17β (E2: 0.1 μg mL–1) or FSH on in vitro maturation (IVM) of porcine oocytes derived from small follicles (SF: 1–2 mm in diameter). The COC were aspirated from MF of porcine ovaries obtained at slaughterhouse and cultured for IVM in mPOM (with 1 mM dibutyryl cAMP, 10 IU mL–1 of eCG, and 10 IU mL–1 of hCG for 20 h and then without those for 24 h in an atmosphere of 5% CO2 in air at 39°C) after washing 3 times. The COC from SF, which were aspirated at the same time with COC from MF, were precultured in the absence or presence of E2 or E2 plus FSH for 6 h before IVM culture. After the culture, oocytes were denuded from cumulus cells with 0.1% (vol/vol) hyaluronidase and the meiotic stage was observed. Relative transcript abundance of FSH and EGF receptors of CC was also examined by real-time RT–PCR just after preincubation for 6 h. Statistical analysis of data from 3 to 5 replicates was analysed by ANOVA and Tukey's multiple comparison tests. Maturation rate of oocytes from SF (40.6 ± 3.1%) was significantly lower than that of oocytes from MF controls (78.8 ± 2.8%, P &lt; 0.01). Preincubation in the presence of E2 alone and E2 plus 0.005 IU of FSH significantly increases the maturation rate of oocytes from SF (56.8 ± 1.5 and 55.7 ± 3.1%, respectively, P &lt; 0.01), although the rate was still lower than MF controls. However, in the presence of E2 plus a higher concentration of FSH (0.05 and 0.5 IU), oocyte maturation rate was similar (36.3 ± 2.4 and 33.7 ± 1.9%, respectively) to SF controls and lower than those of E2 alone and E2 plus 0.005 IU of FSH groups. Relative transcript abundance of FSH receptor of CC increased (P &lt; 0.01) during preincubation in the presence of E2, but decreased in the presence of 0.05 IU of FSH. There were no significant differences in the transcript abundance of EGF receptors among treatments during preincubation (P = 0.09). In conclusion, preincubation of COC from SF in the presence of E2 alone and E2 plus 0.005 IU of FSH improves the maturation rate of the oocytes, whereas the presence of FSH more than 0.05 IU mL–1 concealed the positive effect. These effects may be yielded by change in the relative transcript abundance of FSH receptor of COC through the treatments.

Diminished ovarian reserve (DOR) in the US ART population: diagnostic trends among 183,555 cycles from the society for assisted reproductive technology – clinical outcomes reporting system (SART-CORS)

DOR diagnosis is on the rise despite vaguely defined diagnostic criteria. Possible explanations include the following: a trend towards older patients seeking ART, additional diagnostic modalities such as antral follicle count (AFC) and anti-mullerian hormone (AMH), and clinics’ desire to attract patients and/or explain suboptimal success rates. DOR has generally been associated with poorer ART outcomes, and over-diagnosis causes unnecessary distress to patients. ESHRE defined poor ovarian response (POR) as cycle cancellation for poor response or fewer than four oocytes at retrieval following conventional gonadotropin stimulation (>149 IU FSH daily)1.