There was no specific funding for the preparation of these proposed guidelines. Meetings were held opportunistically during scientific conferences and using online communication tools. H.N.C. is a scientific consultant for ESCO, supplier of Miri TL. I.E.A. is a minor shareholder in Unisense Fertilitech, supplier of the EmbryoScope. Full disclosures of all participants are presented herein. The remaining authors have no conflict of interest.

Multinucleation in two-cell-stage embryos is a frequent event, which is reversible in a high proportion of embryos, without impact on the implantation rate; and embryos with multinucleated blastomeres have a reduced outcome compared with those with binucleated blastomeres when multinucleation is present in four-cell-stage embryos.

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Abnormal luteal function is a common issue in assisted reproduction techniques associated with ovarian stimulation probably due to low levels of LH in the middle and in the late luteal phase. This defect seems to be associated with supraphysiological steroid levels at the end of follicular phase. The luteal phase insufficiency has not got a diagnostic test which has proven reliable in a clinical setting. Luteal phase after ovarian stimulation becomes shorter and insufficient, resulting in lower pregnancy rates. Luteal phase support with progesterone or hCG improves pregnancy outcomes and no differences are found among different routes of administration. However, hCG increases the risk of ovarian hyperstimulation syndrome. In relation to the length of luteal support, the day of starting it remains controversial and it does not seem necessary to continue once a pregnancy has been established. After GnRHa triggering ovulation, intensive luteal support or hCG bolus can overcome the defect in luteal phase, but more studies are needed to show the LH utility as support.

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The aim of the present study was to compare the levels of 17β-estradiol, testosterone, progesterone, inibin, and nitric oxide in follicular fluid between superovulated mares (G1) using equine pituitary extract (EPE) and nonsuperovulated mares (G2) used as control. Two estrus cycles from 24 mares (G1 = 12; G2 = 12) were monitored. In G1, the first cycle was used to determine the ovulation day (Day 0), followed by the administration of PGF2 (250 μg daily of cloprostenol; Sincrocio™, Ouro Fino, São Paulo, Brazil) on the first and second day of treatment with EPE. During the second cycle, EPE (25 mg i.m.) was administered twice daily from Day 7 to the ultrasound detection of a majority of follicles at least 35 mm in diameter. At this time, 2500 IU of hCG IV (Vetecor™, Hertape Calier, Minas Gerais, Brazil) was given. The same protocol was used for G2 as was used for G1 except that EPE administration was replaced by saline injections. For follicular aspiration, the mares were sedated with xilazine (0.5 mg kg-1; Sedazine™, Fort Dodge, São Paulo, Brazil) and acepromazine (0.05 mg kg-1; Acepram 1%™, Univet, Milton, Ontario Canada); hioscin bromide (Buscofin™, Agener União, São Paulo, Brazil) was also administered to reduce intestinal motility and to make ovary manipulation by way of the rectum easier. All follicles larger than 35 mm were aspirated using a Cook® double lumen needle (12 GA) in a transvaginal aspiration probe guided by ultrasonography. The number of aspirated preovulatory follicles was 4.75 ± 2.7 and 1.0 ± 0.0 for G1 and G2, respectively. The follicular fluid collected was centrifuged at 600 × g for 10 min in order to separate the fluid from the cells. The fluid was stored at -20°C and sent to the University of California (Davis, USA) for hormonal assay. The nitric oxide dosage was determined at São Paulo State University (Botucatu, Brazil) using the luminescent reaction of Griess. The comparison between groups of hormonal levels and nitric oxide was performed using the t-test at 5% significance. The results of the present study showed that the EPE treatment (G1), under the experimental conditions, did not lead to a significant change in the mean concentrations of the evaluated hormones or of nitric oxide in the follicular fluid when compared with the levels in G2. However, differences were found among the individuals only in G1 in all hormones (P < 0.05), with exception of progesterone (P > 0.05). Table 1.Mean and standard error of the follicular fluid hormones and nitric oxide levels of groups G1 and G2 Supported by FAPESP, Brazil.

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