A Review on the Reproductive Dysfunction in Farmed Finfish

Globally, fish production in the wild is decreasing, and different aquaculture systems are presently being used for broodstock development in the captivity. Seasonally, broodstock raised in captivity exhibit different form of reproductive dysfunction at the level of the brain-pituitary-gonad (BPG) axis. Primarily, vitellogenic completion and final oocyte maturation are inhibited in females, and males fail to spermiate spontaneously in the captivity. Reproductive dysfunctions are also observed during sexual differentiation, pubertal onset and sex conversion periods in teleosts. To overcome these problems, different hormonal preparations, primarily gonadotropin-releasing hormones (GnRH) are used. In recent years, kisspeptins have been shown to be potent in inducing gonadal growth and maturation in teleost fish. Understanding the form of reproductive dysfunction is important in formulating suitable hormonal preparations for inducing gametogenesis. The paper reviews the problem of reproductive dysfunction and their possible reason for formulating different hormonal preparations.

GnRH-Agonist Ovulation Trigger in Patients Undergoing Controlled Ovarian Hyperstimulation for IVF with Stop GnRH-Agonist Combined with Multidose GnRH-Antagonist Protocol

<b><i>Objective:</i></b> This study aimed to characterize those patients undergoing the stop gonadotropin-releasing hormone (GnRH)-agonist combined with multidose GnRH-antagonist protocol, with suboptimal response to GnRH-agonist trigger in in vitro fertilization (IVF) cycles. <b><i>Design:</i></b> This is a cohort study. <b><i>Setting:</i></b> The study was conducted in a university hospital. <b><i>Patients:</i></b> All consecutive women admitted to our IVF unit from February 2020 through November 2020 who reached the ovum pick-up stage were reviewed. <b><i>Interventions:</i></b> Triggering final oocyte maturation by GnRH-ag alone (GnRH-ag trigger group), or combined with hCG (dual trigger group), in patients undergoing the stop GnRH-agonist combined with multidose GnRH-antagonist protocol was performed. <b><i>Main Outcome Measure:</i></b> The main outcome measure was LH level 12 h after the trigger. <b><i>Results:</i></b> Five out of the 32 patients (15.6%) demonstrated suboptimal response as reflected by LH levels &#x3c;15 IU/L 12 h after GnRH-agonist trigger. Moreover, while no differences were observed in oocyte recovery rate, maturity, or embryo quality between the different study groups (GnRH-ag trigger and dual trigger groups), those achieving a suboptimal response to the GnRH-agonist trigger (post-trigger LH &#x3c;15 mIU/mL) demonstrated significantly higher number of follicles and peak estradiol levels at the day of trigger, compared to those with optimal response (post-trigger LH &#x3e;15 mIU/mL). <b><i>Conclusions:</i></b> The stop GnRH-agonist combined with GnRH-antagonist protocol enables the substitution of hCG with GnRH-ag for final oocyte maturation. However, caution should be taken in high responders, where the dual trigger with small doses of hCG (1,000–1,500 IU) should be considered, aiming to avoid suboptimal response (post-trigger LH levels &#x3c;15 IU/L).

P–685 Luteinization after final oocyte maturation induction is not compromised in women that receive double dose of Gonadotrophin-releasing hormone antagonists during controlled ovarian hyperstimulation

Study question Does the use of double dose of GnRH antagonists during COH in women with risk of premature LH surge alter luteinization after final oocyte maturation induction? Summary answer The use of double dose of GnRH antagonist in women with risk of premature luteinizing hormone surge dosent affect luteinization after final oocyte maturation induction. What is known already GnRH antagonists are used to prevent a premature LH surge during controlled ovarian hyperstimulation. The antagonists directly inhibit gonadotrophin release within several hours through competitive binding to pituitary GnRH receptors, producing a rapid suppression of LH and FSH, with no initial flare effect. In women with diminished ovarian reserve (DOR) it is not uncommon that premature luteinization cannot be completely prevented using a daily dose GnRH antagonist. To date, no study has evaluated the effects of using a daily double dose of GnRH antagonists to prevent a premature LH surge and its effect on luteinization after final oocyte maturation induction. Study design, size, duration This monocentric retrospective analysis evaluated the effect on luteinization after final oocyte maturation induction in twenty women during COH who received a daily double dose of GnRH antagonists (Cetrotide 0.25 mg/mL, Merck) from January 2020 to December 2020. Participants/materials, setting, methods Women with severe DOR and history of premature luteinization during COH received a double dose of GnRH antagonist when the leading follicle reached 12–14 mm (am and pm). When two follicles reached ≥18 mm in diameter, final oocyte maturation was induced with dual trigger using Leuprolide acetate and hCG. Progesterone, estradiol, bHCG, and LH levels were measured the day after final oocyte maturation induction to assure adequate luteinization. Main results and the role of chance In total twenty women were included in the analysis. Mean age 36.8± 4.2, AMH 0.65± 0.32 ng/ml, baseline antral follicle count 4± 2.3, serum hormone levels the day of ovulation induction trigger: progesterone 0.89± 0.34 ng/ml, LH 1.6± 2.1 ng/ml, estradiol 1235 ± 1420 pg/ml. Post-surge serum hormone levels average reached adequate levels: estradiol 1645 ± 1116 pg/ml, progesterone 20.4 ±2.2 ng/ml, LH 62.66± 10.5 IU/ml and, bHCG 247±115 IU/ml. A total of 76 oocytes were retrieved (3.8± 0.8 oocytes per patient), 63.1% (48/76) MII, 22% (17/76) MI, 14% (11/76) GV. Limitations, reasons for caution The retrospective nature of the study, small sample size, and potential variability in the study center’s laboratory protocol(s) compared to other reproductive treatment centers may limit the external validity of our findings. Wider implications of the findings: The daily use of double dose of GnRH antagonists during COH offers the possibility of preventing a premature LH surge in women with DOR with high risk of early ovulation, without compromising luteinization after final oocyte maturation induction. Trial registration number NA

P-681 Will the hCG trigger dose used for final oocyte maturation in IVF impact endogenous progesterone during the luteal phase? - A randomized controlled trial

Study question Is there an association between the hCG dose used for ovulation trigger and the endogenous progesterone production during the luteal phase? Summary answer Increased hCG dosing significantly increased the endogenous progesterone level during the luteal phase. What is known already During the luteal phase of an IVF treatment, the endogenous progesterone (P4) production is negatively impacted due to reduced circulating endogenous LH, caused by negative feed-back of elevated steroids; thus, luteal phase support (LPS) with exogenous P4 remains mandatory in IVF. Apart from inducing final oocyte maturation, the gold standard HCG trigger also functions as an early LPS, boosting P4 production by the corpora lutea (CL). P4 plays a pivotal role for embryo implantation and pregnancy, and an optimal P4 level around peri-implantation seems to be essential for the reproductive outcomes of fresh and frozen/thaw embryo transfer cycles. Study design, size, duration A randomized controlled 4-arm study, including a total of 127 IVF patients, enrolled from January 2015 until September 2019 at the Fertility Clinic, Odense University Hospital, Denmark. Participants/materials, setting, methods IVF patients with ≤ 11 follicles ≥ 12 mm were randomized to four groups. Groups 1-3 were triggered with: 5.000 IU, 6.500 IU or 10.000 IU, hCG, respectively, receiving a LPS consisting of 17-α-hydroxy-progesterone (17α OH P4) to distinguish the endogenous P4 from the exogenous supplementation. Group 4 (control) was randomized to a 6.500 IU hCG trigger and standard LPS. A total of eight blood samples were drawn during the early luteal phase. Main results and the role of chance A total of 94 patients completed the study: 21, 22, 25 and 26 patients in each group, respectively. Baseline characteristics were similar, except for the endogenous LH level and cycle lengths. There were no significant differences between groups regarding ovarian stimulation, number of oocytes and embryos. The median number of follicles ≥ 12mm on the day of trigger was 8.5, resulting in 6.6 oocytes being retrieved. Significant differences in P4 levels were seen at OPU+8 (p &lt; 0.001), OPU+10 (p &lt; 0.001) and OPU+14 (p &lt; 0.001), with positive correlations between P4 level and hCG dose. Groups compared individually showed significant difference in P4 between low and high trigger dose at OPU+4 group 1 and 3 (p = 0.037) and OPU+8 group 1 and 3 (p = 0.007) and between all the three groups around implantation at OPU+6 group 1 and 2 (p = 0.011), group 2 and 3 (p = 0.042) and group 1 and 3 (p &lt; 0.001). Higher P4 levels around implantation were related to follicle count and to pregnancy. After logistic regression analyses there were still significant individual differences between the groups. Limitations, reasons for caution Although patients were randomized and strict inclusion and exclusion criteria were used, the RCT was un-blinded, including a relatively small number of patients. Moreover, for dosing purposes urinary hCG as well as recombinant hCG was used and pharmacokinetics differ. Finally, the P4 level could be influenced by circadian fluctuations. Wider implications of the findings This is the first study to explore dose-responses in circulating P4 after hCG trigger in IVF patients. Increasing the hCG trigger dose increased the endogenous P4 around peri-implantation. Personalizing the hCG trigger dose could be a key point to secure the most optimal P4 mid-luteal phase P4 level. Trial registration number Eudract 2013-003304-39

P–663 The ratio of serum progesterone (P4) to the number of follicles (P4/Follicle) is a more objective parameter for euploidy rate as compared to systemic progesterone

Study question Does the ratio of serum progesterone (P4) to the number of follicles (P4/Follicle) on the day of final oocyte maturation affect the ploidy status of the embryos? Summary answer A high P4/Follicle ratio negatively affects the euploid rate of the embryos. What is known already During ovarian stimulation, exogenous gonadotropins are administered to achieve multifollicular growth. Intense gonadotropin stimulation towards the end of the follicular phase seems to cause a premature progesterone rise in stimulated IVF cycles. The impact of serum progesterone elevation during the follicular phase has been studied intensively. Though most studies have focused on the effect of progesterone elevation on the endometrial receptivity, little is known about its possible impact on embryo development and ploidy status. The only study that investigated the effect of progesterone on the embryo ploidy status, was unable to show any significant impact. Study design, size, duration This retrospective study was performed at ART Fertility Clinics Abu Dhabi, UAE and Muscat, Oman. All stimulation cycles (n = 975) were performed between January 2015 to December 2019 with patients aged between 18–45, Body mass index (BMI) of 18–35, stimulated either with rFSH or hMG. All embryos underwent ICSI and Preimplantation Genetic Testing for Aneuploidies (PGT-A),Patients with surgical sperm extraction, warmed oocytes or natural cycle IVF were excluded. Participants/materials, setting, methods Serum P4 was measured on the last ultrasound prior triggering for final oocyte maturation. The P4/Follicle ratio was calculated as the ratio of P4 on trigger day to the number of follicles &gt; 10 mm on the last ultrasound. Serum P4 and P4/Follicle ratio were then analyzed using linear and univariate regression model to find potential correlation with the number of oocytes retrieved, number of mature oocytes, embryo quality (day 3 and 5), and euploid rate. Main results and the role of chance A total of 975 cycles were analyzed, with a mean age of 33.88±0.05 years, a mean BMI of 26.7±0.035 kg/m2. The mean number of oocytes collected was 12.53±0.058. Mean serum P4 on trigger day was 0.83±0.005 ng/ml and higher serum P4 values were observed as the number of oocytes retrieved and the number of mature oocytes increased (β = 0.026, p &lt; 0.0001 and β = 0.028, p &lt; 0.001, respectively). On the other hand, the mean P4/Follicle ratio was 0.056±0.00041 ng/ml and, unlike serum P4, the P4/Follicle ratio showed a negative correlation with the number of oocytes retrieved as well as with the number of mature oocytes (β=–0.001, p &lt; 0.001 and β=–0.001, p &lt; 0.001, respectively). While day 3 embryos were not affected by serum P4 or P4/Follicle ratio, the blastocyst quality was negatively affected by both increasing serum P4 levels and the P4/Follicle ratio (β=–0.012 p &lt; 0.05, β=–0.002, p &lt; 0.001, respectively). Euploid rates were positively correlated in cycles with increased serum P4 β = 0.18, p &lt; 0.001), while negatively correlated in cycles with a high P4/Follicle ratio (β=–0.015, p &lt; 0.001). After adjusting for potential confounders, only P4/Follicle remained as a significant negative factor for euploid rate (β=–0.004, p &lt; 0.001, 95% CI: –0.007- –0.001, p &lt; 0.001), which was not observed for serum P4 (p = 0.46). Limitations, reasons for caution This is an observational study based on retrospective data; an improved extrapolation of the results might be obtained by performing a prospective study. Wider implications of the findings: The findings of this study should encourage clinicians to optimize the ovarian stimulation protocols not only based on serum P4, but also considering the P4/Follicle ratio. Trial registration number Not applicable

P–698 Antimüllerian Hormone (AMH) value as a predictive marker of cycle ploidy outcome

Study question Do woman with diminished ovarian reserve exhibit poor blastocyst formation and ploidy outcomes, irrespective of age? Summary answer Patients with extreme diminished ovarian reserve (AMH≤0.65ng/ml) have a lower chance to have at least one euploid blastocyst compared to their age-related reference population (AMH=1.3–6.25ng/ml). What is known already AMH is an established marker of the ovarian reserve for predicting ovarian response to ovarian stimulation and it is strongly correlated with female age. However, it has been suggested that AMH is not only a quantitative, but also a qualitative biomarker of oocyte/embryo competence. Previous studies show conflicting outcomes as to whether reduced ovarian reserve per se is associated with decreased oocyte developmental competence, leading to increased aneuploidy rates in embryos independent of the patient’s age. Study design, size, duration A retrospective analysis was performed between March 2017 and July 2020 at ART Fertility Clinics (Abu Dhabi) including all couples that were triggered for final oocyte maturation and planned for Preimplantation Genetic Testing for Aneuploidies (PGT-A). Patients were stratified into four age categories [≤30, 31–35, 36–40, &gt;40 years]. For each age category patients were further divided into three AMH groups: ≤0.65ng/ml, 0.65–1.3ng/ml and 1.31–6.25ng/ml (reference group). Participants/materials, setting, methods Trophectoderm biopsy samples were subjected to Next Generation Sequencing. AMH serum levels (ng/ml) were determined using the commercial fully automated Elecsys® (Roche) assay. Patients with a Progesterone rise of &gt; 1.5ng/ml on the day of final oocyte maturation and patients with AMH values &gt;6.25ng/ml were excluded from the analysis. Per patient that was triggered, the chance to have at least one euploid blastocyst in that cycle, was calculated. Main results and the role of chance A total of 1.300 couples were included with an mean maternal age of 35.6±6.2 years, AMH of 2.1 ±1.5ng/ml and body mass index of 27.5±5.0 kg/m2. The chance to have at least one blastocyst biopsied per cycle was affected in all patients with extreme low AMH (≤0.65ng/ml), irrespective of age; ≤30 years: 58.33%–100.00%–94.84% (p &lt; 0.001); 31–35 years: 50.00%–74.55%–95.32% (p &lt; 0.001); 36–40 years: 56.52%–81.93%–92.56% (p &lt; 0.001) and ≥40 years: 38.06%–73.02%–88.24% (p &lt; 0.001), for AMH ≤0.65ng/ml, 0.65–1.3ng/ml and 1.31–6.25ng/ml, respectively. In all age categories, patients with AMH values ≤0.65ng/ml had a significantly reduced probability of having a euploid blastocyst compared to the reference group (1.31–6.25ng/ml). For women ≤30 years the chances of getting a euploid blastocyst decreased from 88.89% (n = 252) to 41.67% (n = 12) (OR 0.01 [0.03–0.30], p &lt; 0.001), for 31–35 years from 88.09% (n = 235) to 43.75% (n = 32) (OR 0.10 [0.05–0.23], p &lt; 0.001), for 36–40 years from 77.67% (n = 215) to 21.74% (n = 69) (OR 0.08 [0.04–0.15], p &lt; 0.001) and among women &gt;40 years from 29.42% (n = 102) to 6.45% (n = 155) (OR 0.16 [0.08–0–36], p &lt; 0.001). Woman within AMH range of 0.65–1.3ng/ml presented the same decreased probability of having a euploid blastocyst only when 31–35 (52.73%, n = 55) or 36–40 years old (56.63%, n = 83) (OR 0.15 [0.08–0.29], p &lt; 0.001 and OR 0.37 [0.22–0.64], p &lt; 0.001, respectively). Limitations, reasons for caution The main limitation of this study is its retrospective design. Wider implications of the findings: AMH is a clear biomarker of oocyte-embryo competence. Incorporation of AMH-specific counseling recommendations into clinical practice guidelines, could lead to a more informed guidance on cycle ploidy outcomes, rather than age alone. Trial registration number Not applicable

P–681 Will the hCG trigger dose used for final oocyte maturation in IVF impact endogenous progesterone during the luteal phase? - A randomized controlled trial

Study question Is there an association between the hCG dose used for ovulation trigger and the endogenous progesterone production during the luteal phase? Summary answer Increased hCG dosing significantly increased the endogenous progesterone level during the luteal phase. What is known already During the luteal phase of an IVF treatment, the endogenous progesterone (P4) production is negatively impacted due to reduced circulating endogenous LH, caused by negative feed-back of elevated steroids; thus, luteal phase support (LPS) with exogenous P4 remains mandatory in IVF. Apart from inducing final oocyte maturation, the gold standard HCG trigger also functions as an early LPS, boosting P4 production by the corpora lutea (CL). P4 plays a pivotal role for embryo implantation and pregnancy, and an optimal P4 level around peri-implantation seems to be essential for the reproductive outcomes of fresh and frozen/thaw embryo transfer cycles. Study design, size, duration A randomized controlled 4-arm study, including a total of 127 IVF patients, enrolled from January 2015 until September 2019 at the Fertility Clinic, Odense University Hospital, Denmark. Participants/materials, setting, methods IVF patients with ≤ 11 follicles ≥ 12 mm were randomized to four groups. Groups 1–3 were triggered with: 5.000 IU, 6.500 IU or 10.000 IU, hCG, respectively, receiving a LPS consisting of 17-α-hydroxy-progesterone (17α OH P4) to distinguish the endogenous P4 from the exogenous supplementation. Group 4 (control) was randomized to a 6.500 IU hCG trigger and standard LPS. A total of eight blood samples were drawn during the early luteal phase. Main results and the role of chance A total of 94 patients completed the study: 21, 22, 25 and 26 patients in each group, respectively. Baseline characteristics were similar, except for the endogenous LH level and cycle lengths. There were no significant differences between groups regarding ovarian stimulation, number of oocytes and embryos. The median number of follicles ≥ 12mm on the day of trigger was 8.5, resulting in 6.6 oocytes being retrieved. Significant differences in P4 levels were seen at OPU+8 (p &lt; 0.001), OPU+10 (p &lt; 0.001) and OPU+14 (p &lt; 0.001), with positive correlations between P4 level and hCG dose. Groups compared individually showed significant difference in P4 between low and high trigger dose at OPU+4 group 1 and 3 (p = 0.037) and OPU+8 group 1 and 3 (p = 0.007) and between all the three groups around implantation at OPU+6 group 1 and 2 (p = 0.011), group 2 and 3 (p = 0.042) and group 1 and 3 (p &lt; 0.001). Higher P4 levels around implantation were related to follicle count and to pregnancy. After logistic regression analyses there were still significant individual differences between the groups. Limitations, reasons for caution Although patients were randomized and strict inclusion and exclusion criteria were used, the RCT was un-blinded, including a relatively small number of patients. Moreover, for dosing purposes urinary hCG as well as recombinant hCG was used and pharmacokinetics differ. Finally, the P4 level could be influenced by circadian fluctuations. Wider implications of the findings: This is the first study to explore dose-responses in circulating P4 after hCG trigger in IVF patients. Increasing the hCG trigger dose increased the endogenous P4 around peri-implantation. Personalizing the hCG trigger dose could be a key point to secure the most optimal P4 mid-luteal phase P4 level. Trial registration number Eudract 2013–003304–39

Effect of a low water salinity on oocyte maturation, oocyte hydration, ovulation and egg quality in rainbow trout.

Water salinity is an important environmental factor known to have detrimental effects on salmonid reproduction, mostly when migrating female broodfish are held in sea water. In contrast, data obtained in freshwater are scarce and the impact of low water salinity during reproduction in freshwater is currently unknown. For this reason, and because ion and water fluxes are critical for the final steps of the female gamete formation, including oocyte hydration and ovulation, the aim of the present study was to investigate the impact of low salinity water on final oocyte maturation, ovulation and, ultimately, on egg quality, using rainbow trout as a physiological model and relevant aquaculture species. Fish from the same commercial strain were raised throughout their lifecycle either in a site characterized by low concentrations of Na+, K+, and Cl- ions in the water or in a closely located control site exhibiting standard salinity levels. Egg quality and duration of final oocyte maturation were investigated using innovative phenotyping tools such as automatic assessment of egg viability using the VisEgg system and non-invasive echograph-based monitoring of final oocyte maturation duration, respectively. Oocyte hydration during final oocyte maturation and after ovulation was also investigated. Finally, molecular phenotyping was performed using real-time PCR-based monitoring of several key players of final oocyte maturation and ovulation associated with ion and water transport, inflammation, proteolytic activity, and coagulation. Oocyte hydration and gene expression data were analyzed in the light of the duration of final oocyte maturation. Here we show that low water salinity negatively influences final oocyte maturation, ovulation and, ultimately, egg quality. Low water salinity triggers delayed ovulation and lower oocyte viability. When investigating the impact of low water salinity on final oocyte maturation duration, individuals presenting the most severe phenotypes exhibited impaired oocyte hydration and abnormally reduced gene expression levels of several key players of the ovulatory process. While the under expression of water (e.g., aquaporins) and ion (e.g., solute carriers) transporters is consistent with impaired oocyte hydration, our observations also indicate that the entire ovulatory gene expression program is disrupted. Our results raise the question of the mechanisms underlying the negative influence of low salinity water on the dynamics of the preovulatory phase, on the control of the oocyte homeostasis, including hydration, and on the overall success of the maturation-ovulation process.