Clinical Efficacy of Follitropin Alfa in GnRH-Antagonist Protocols: A Prospective Observational Phase IV Study on the Use of Biosimilar Follitropin Alfa r-hFSH in Assisted Reproductive Technology in a Routine Care Setting

Background: This phase IV routine care study evaluated ovarian responses when using a biosimilar follitropin alfa r-hFSH (Bemfola®) for controlled ovarian stimulation (COS) in women undergoing assisted reproductive technology (ART) treatment who were pituitary-suppressed with a gonadotrophin-releasing hormone (GnRH) antagonist. Methods: This multicenter, prospective, non-comparative, non-interventional study (Germany/Austria) was conducted with 885 women (Mean age of 34.0±4.4 years) for whom COS with Bemfola® and GnRH-antagonist for pituitary suppression were applied as part of in vitro fertilization (IVF) treatment with/without intracytoplasmic sperm injection (ICSI) observing routine clinical-practice protocols. Primary endpoint was the number of retrieved cumulus-oocyte-complexes (COCs). Results: Among 986 ART cycles, COS was given for 9.9±1.8 days (First-day r-hFSH dose of 220.7±68.9 IU; mean total dose of 2184.3±837.5 IU). It was revealed that 99.1% of cycles resulted in follicular puncture, with mean of 10.7±6.6 oocytes retrieved. Successful fertilization took place after IVF/ICSI in 93.8% of follicular punctures. Freeze-all was performed in 14.2% of cycles. Fresh embryo transfer was performed in 76.9% of cycles with follicular puncture; mean day of transfer was 3.5±1.3 and average number of transferred embryos was 1.76±0.50. Clinical pregnancy rate was 30.2% of embryo-transfer cycles and 23.4% of started cycles. Sixty-nine reports of ovarian hy-perstimulation syndrome (7.0% of started cycles) were documented. Conclusion: COS with Bemfola® in GnRH-antagonist IVF/ICSI protocols in a routine care setting led to an appropriate ovarian response allowing oocyte retrieval in 99.1% of initiated cases.

Anesthesia parameters in in vitro fertilization

The review describes certain practical aspects of follicular puncture anesthesia. It presents data on the penetration of anesthetics and other drugs used during anesthesia into the follicular fluid as well as the effect of certain drugs and conditions on the reproductive outcome of treatment. Various options for anesthesia are described that can be used depending on the characteristics of the patient's psychoemotional state and the number of punctured follicles.

ZP1 mutations are associated with empty follicle syndrome: evidence for the existence of an intact oocyte and a zona pellucida in follicles up to the early antral stage. A case report.

Empty follicle syndrome (EFS) is the complete failure to retrieve oocytes after ovarian stimulation. Although LHCGR and ZP3 were identified as causative genes, it is still unclear what happens to these patients’ oocytes, and the pathogenesis of EFS remains obscure. Here, we identified six novel ZP1 mutations associated with EFS and female infertility that was inherited recessively in five unrelated families. Studies in CHO-K1 cells showed that these mutations resulted in either degradation or truncation of ZP1 protein. Immunohistochemistry using ovarian serial sections demonstrated that all preantral follicles had normal architecture, but with a thin ZP, lacking ZP1, surrounding the growing oocytes. The antral follicles were also defective in normal cumulus–oocyte complex organisation, leading us to speculate that the lack of ZP1 might lead to oocyte degeneration or increased fragility of the oocyte during follicular puncture, ultimately resulting in EFS. To our knowledge, this is the first study that presents morphological evidence showing normal preantral folliculogenesis with abnormal ZP assembly in EFS patients. Our data provides a better understanding of the biological functions of ZP1 in human ZP assembly and folliculogenesis and gives new insights into the pathogenesis of EFS and possible therapeutic developments.

Impact of Melatonin Supplementation in Women with Unexplained Infertility Undergoing Fertility Treatment

Unexplained infertility occurs when common causes for a couple’s inability to conceive have been excluded. Although origins of idiopathic infertility are still unclear, factors, such as an altered oxidative balance, are believed to be involved. Melatonin is an outstanding antioxidant reportedly present in the follicular fluid (FF), which has been suggested as a useful tool in the management of human fertility. Herein, we observed that intrafollicular concentrations of melatonin were blunted in women with unexplained infertility (UI), which was associated with a marked oxidative imbalance in UI patients’ FF. Based on these findings, this randomized pilot study was aimed at assessing whether exogenous melatonin ameliorated oxidative stress and improved in vitro fertilization (IVF) success rates in UI. Thus, 3 mg/day or 6 mg/day of melatonin were given to UI patients for a period spanning from the first appointment to control ovarian stimulation until the day of follicular puncture. Our results indicate that melatonin supplementation, irrespective of the two doses tested, ameliorated intrafollicular oxidative balance and oocyte quality in UI patients, and that this translated into a slight increase in the rate of pregnancies/live births. Therefore, although the indoleamine has shown therapeutic potential in this clinical setting, larger clinical trials in populations with different backgrounds are encouraged to corroborate the usefulness of melatonin.

Cryopreservation of Bălțată cu Negru Românească Cattle Immature Oocytes by Slow Freezing Method: Preliminary Research

Research on bovine oocytes cryopreservation is important for successful preservation of genetically valuable animal. The transvaginal ultrasound-guided follicular puncture coupled with in vitro embryo production has become competitive and alternative method for MOET (Multiple ovulation and embryo transfer) in dairy cattle. The aim of this preliminary research is to presents the result of Bălțată cu Negru Românească (BNR) cows oocytes recovery by two different protocols and its cryopreservation by slow freezing method. By applying the recovery oocytes from slaughterhouse ovary we obtained an average of 16.34 ± 6.71 oocytes per cow, much higher compared with the Ovum Pick Up (OPU) method, which reveals an average of 2.75 ± 0.2 oocytes per cow. After applying the slow freezing procedures using the ethylene glycol cryoprotectant we observed the oocytes with cumulus cells normal with the spherical shape and normal zone pellucida.

Research Resource: Preovulatory LH Surge Effects on Follicular Theca and Granulosa Transcriptomes

The molecular mechanisms that regulate the pivotal transformation processes observed in the follicular wall following the preovulatory LH surge, are still not established, particularly for cells of the thecal layer. To elucidate thecal cell (TC) and granulosa cell (GC) type-specific biologic functions and signaling pathways, large dominant bovine follicles were collected before and 21 hours after an exogenous GnRH-induced LH surge. Antral GCs (aGCs; aspirated by follicular puncture) and membrane-associated GCs (mGCs; scraped from the follicular wall) were compared with TC expression profiles determined by mRNA microarrays. Of the approximately 11 000 total genes expressed in the periovulatory follicle, only 2% of thecal vs 25% of the granulosa genes changed in response to the LH surge. The majority of the 203 LH-regulated thecal genes were also LH regulated in GCs, leaving a total of 57 genes as LH-regulated TC-specific genes. Of the 57 thecal-specific LH-regulated genes, 74% were down-regulated including CYP17A1 and NR5A1, whereas most other genes are being identified for the first time within theca. Many of the newly identified up-regulated thecal genes (eg, PTX3, RND3, PPP4R4) were also up-regulated in granulosa. Minimal expression differences were observed between aGCs and mGCs; however, transcripts encoding extracellular proteins (NID2) and matrix modulators (ADAMTS1, SASH1) dominated these differences. We also identified large numbers of unknown LH-regulated GC genes and discuss their putative roles in ovarian function. This Research Resource provides an easy-to-access global evaluation of LH regulation in TCs and GCs that implicates numerous molecular pathways heretofore unknown within the follicle.

Superovulation following follicular synchronization with GnRH at random stages of the oestrous cycle in heifers

The objective of this study was to develop a superovulatory program based on synchronization of follicular waves with GnRH which could be applied regardless of the stage of the oestrous cycle. 36 heifers were subjected to this experiment and GnRH (Cystorelin, 200 µg) was applied between Days 0 and 7 (n = 15), 8 and 12 (n = 8) or 13 and 20 (n = 13) of the oestrous cycle. Four days after GnRH treatment, all follicles ≥ 6 mm of heifers (n) were either punctured (n = 21) or left intact (n = 15). All heifers were superstimulated from Day 6 to Day 10 after GnRH treatment with 320 mg Folltropin-V. In parallel, 21 heifers were superstimulated in a conventional manner (Days 8 to 12) and were used as controls. The homogeneity of follicular inventories among Stage-groups occurred within 4 days of GnRH treatment for follicles ≥ 7 mm but only 2 days after follicular puncture for follicles 4 to 6 mm. In response to the follicular puncture, the mean number of follicles 4 to 6 mm increased in heifers of the punctured group (P < 0.01). Following the superstimulation, the follicular (P < 0.01) and ovulatory (P < 0.01) responses were higher in the punctured group than in the nonpunctured group. The in vivo production of transferable embryos in the punctured group was similar to that of the nonpunctured group but it was lower (P < 0.01) than in heifers of the control group. In conclusion, results from the present study indicate that regardless of the stage of the oestrous cycle, the homogeneity of follicular inventories following the follicular synchronization is obtained using GnRH treatment and follicular puncture. The in vivo production of embryos was severely compromised in the present study with heifers. Causes of such reduction in the in vivo production of embryos are still unknown.

Superovulation following follicular synchronization with GnRH at random stages of the oestrous cycle in heifers: oocyte competence and in vitro embryo production

The objective of this study was to develop a superovulatory program based on the synchronization of follicular waves with GnRH which could be applied regardless of the stage of the oestrous cycle. In this experiment, GnRH was given to 30 heifers in lactation between Days 0 and 7 (n = 13), 8 and 12 (n = 12), 13 and 16 (n = 5) of the oestrous cycle. Twenty-four heifers were used as controls and did not receive any GnRH. All follicles ≥ 6 mm were punctured 4 days after GnRH treatment in treated animals and between Days 8 and 12 of the oestrous cycle in control heifers. Two days after the follicular puncture, all heifers were superstimulated with 160 mg Folltropin-V given twice daily over 2 days. Oocytes were collected 42 h after the last FSH treatment. The oocytes were subjected to IVM/IVF and the developmental competence of embryos was compared. In vitro production of embryos was affected only by the stages of the oestrous cycle when the GnRH treatment was given and not by the GnRH treatment. No difference (P > 0.1) in the mean number of oocytes, cleavage and embryo production was noted between the control animals and the animals treated with GnRH in the late phase of the oestrous cycle. The mean number of blastocysts was higher (P < 0.05) in heifers treated with GnRH in the mid and the late phase of the oestrous cycle than in the early phase. In conclusion, the in vitro production of embryos was compromised in the present study with heifers following the follicular synchronization with GnRH. This procedure is advantageous for the in vitro production of bovine embryos since the spontaneous oestrus is eliminated. However, more investigations are needed to increase the competence of oocytes obtained following this procedure.

274 ASSESSMENT OF TRYPSIN AND ANTIBIOTIC TREATMENT EFFECTIVENESS IN IN VITRO-MATURED OOCYTES EXPERIMENTALLY EXPOSED TO LEPTOSPIRA INTERROGANS SEROVAR GRIPPOTYPHOSA

Techniques of production and transfer of embryos is safe as long as it follows the control regulations defined by the manual of the International Embryo Transfer Society (IETS) for treating oocytes with trypsin, antibiotics, and TCM-199 medium. The aim of this work was to evaluate the effectiveness of treatments, established by IETS, in bovine oocytes experimentally exposed to Leptospira interrogans serovar Grippotyphosa and to assist implementation of quality control standards on in vitro embryo production. The oocytes were obtained through follicular puncture of ovaries derived from the slaughterhouse. They were selected and divided into 4 groups: the control group and groups exposed to 5, 10, and 30 μL of an L. interrogans strain at 4.7 × 105 μL-1; and 4 additional groups exposed to the same concentrations of another L. interrogans strain at 6.3 × 105 μL-1, in which the gene for virulence is not expressed. The groups were kept in maturation medium (TCM-199 medium, 0.5 (iLof FSH, 50IU mL-1 hCG, 1μL mL-1 17-βiestradiol) and incubated at 38°C, 5% CO2, and 95% humidity for 24 h. All the groups were separately subjected to the treatments with antibiotic, trypsin, and TCM-199 medium after maturation. The treatment involved 10 drops (each 200 μL), with 8 drops of TCM-199 medium and 2 drops of antibiotic (penicillin 10000 IU mL-1 and streptomycin 10 mg mL-1) or trypsin 0.25%; exposed to trypsin and antibiotic for 120 s. For the sequential washes, all drops contained TCM-199 medium. The analysis for presence of the pathogen by dark-field optical microscopy showed that in the groups exposed to L. interrogans and subjected to antibiotic washes, the effectiveness was 50% (100/200) for the group exposed to 5 μL, 40% (80/200) for that exposed to 10 μL, and 22.5% (45/200) for that exposed to 30 μL. We found the same results after the trypsin washes. After the washings with TCM-199 medium, the groups infected with 5 and 10 μL presented 100% of effectiveness; however, for the group infected with 30 μL, the washings were not effective. For the groups exposed to L. interrogans that did not express virulence, after the washings with antibiotics as well as with trypsin, the results showed no effectiveness in all of them (n = 200). Yet, after washings with TCM-199, the group exposed to 5 μL showed 28.5% (57/200) of effectiveness, whereas in those exposed to 10 and 30 μL, the medium washes were not effective. Complementary studies are being made with ultramicrotome cut and polymerase chain reaction for more reliable conclusions, to confirm the results. With such results, we conclude that the quality control regulations established by IETS for IVP could be reviewed and possibly redefined, because the effectiveness of the treatment may depend not only on the pathogen species, but also its virulence as well as its concentration and the action of the treatments on the type of pathogen. We thank Vitrocel/Embriolife for supporting the laboratory.