Shear stress in the intervillous space promotes syncytial formation of iPS cells-derived trophoblasts†.

The intervillous space of human placenta is filled with maternal blood, and villous trophoblasts are constantly exposed to the shear stress generated by maternal blood pressure and flow throughout the entire gestation period. However, the effects of shear stress on villous trophoblasts and their biological significance remain unknown. Here, using our recently established naïve human pluripotent stem cells-derived cytotrophoblast stem cells (nCTs) and a device that can apply arbitrary shear stress to cells, we investigated the impact of shear stress on early-stage trophoblasts. After 72 h of exposure to 10 dyn/cm2 shear stress, nCTs became fused and multinuclear, and mRNA expression of the syncytiotrophoblast (ST) markers, such as glial cell missing 1, endogenous retrovirus group W member 1 envelope, chorionic gonadotropin subunit beta 3, syndecan 1, pregnancy specific beta-1-glycoprotein 3, placental growth factor, and solute carrier family 2 member 1 were significantly upregulated compared to static conditions. Immunohistochemistry showed that shear stress increased fusion index, human chorionic gonadotropin secretion, and human placental lactogen secretion. Increased microvilli formation on the surface of nCTs under flow conditions was detected using scanning electron microscopy. Intracellular cyclic adenosine monophosphate significantly increased under flow conditions. Moreover, transcriptome analysis of nCTs subjected to shear stress revealed that shear stress upregulated ST-specific genes and downregulated CT-specific genes. Collectively, these findings indicate that shear stress promotes the differentiation of nCTs into ST.

Assessment of active translation in cumulus-enclosed and denuded oocytes during standard in vitro maturation and early embryo development.

STUDY QUESTION: Which actively translated maternal transcripts are differentially regulated between clinically relevant in vitro and in vivo maturation (IVM) conditions in mouse oocytes and zygotes? SUMMARY ANSWER: Our findings uncovered significant differences in the global transcriptome as well as alterations in the translation of specific transcripts encoding components of energy production, cell cycle regulation, and protein synthesis in oocytes and RNA metabolism in zygotes. WHAT IS KNOWN ALREADY: Properly regulated translation of stored maternal transcripts is a crucial factor for successful development of oocytes and early embryos, particularly due to the transcriptionally silent phase of meiosis. STUDY DESIGN, SIZE, DURATION: This is a basic science study utilizing an ICR mouse model, best suited for studying in vivo maturation. In the treatment group, fully grown germinal vesicle oocytes from stimulated ovaries were in vitro matured to the metaphase II (MII) stage either as denuded without gonadotropins (IVM DO), or as cumulus-oocyte complexes (IVM COC) in the presence of 0.075 IU/ml recombinant FSH (rFSH) and 0.075 IU/ml recombinant hCG (rhCG). To account for changes in developmental competence, IVM COC from non-stimulated ovaries (IVM COC-) were included. In vivo matured MII oocytes (IVO) from stimulated ovaries were used as a control after ovulation triggering with rhCG. To simulate standard IVM conditions, we supplemented media with amino acids, vitamins, and bovine serum albumin. Accordingly, in vitro pronuclear zygotes (IMZ) were generated by IVF from IVM DO, and were compared to in vivo pronuclear zygotes (IVZ). All experiments were performed in quadruplicates with samples collected for both polyribosome fractionation and total transcriptome analysis. Samples were collected over three consecutive months. PARTICIPANTS/MATERIALS, SETTING, METHODS: All ICR mice were bred under legal permission for animal experimentation (no. MZE-24154/2021-18134) obtained from the Ministry of Agriculture of the Czech Republic. Actively translated (polyribosome occupied) maternal transcripts were detected in in vitro and in vivo matured mouse oocytes and zygotes by density gradient ultracentrifugation, followed by RNA isolation and high-throughput RNA sequencing. Bioinformatic analysis was performed and subsequent data validation was done by western blotting, radioactive isotope, and mitotracker dye labelling. MAIN RESULTS AND THE ROLE OF CHANCE: Gene expression analysis of acquired polysome-derived high-throughput RNA sequencing data revealed significant changes (RPKM ≥ 0.2; P ≤ 0.005) in translation between in vitro and in vivo matured oocytes and respectively produced pronuclear zygotes. Surprisingly, the comparison between IVM DO and IVM COC RNA-seq data of both fractionated and total transcriptome showed very few transcripts with more than a 2-fold difference. Data validation by radioactive isotope labelling revealed a decrease in global translation bof20% in IVM DO and COC samples in comparison to IVO samples. Moreover, IVM conditions compromised oocyte energy metabolism, which was demonstrated by both changes in polysome recruitment of each of 13 mt-protein-coding transcripts as well as by validation using mitotracker red staining. LARGE SCALE DATA: The data discussed in this publication have been deposited in NCBI's Gene Expression Omnibus and are accessible through GEO Series accession number GSE241633 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE241633). LIMITATIONS, REASONS FOR CAUTION: It is extremely complicated to achieve in vivo consistency in animal model systems such as porcine or bovine. To achieve a high reproducibility of in vivo stimulations, the ICR mouse model was selected. However, careful interpretation of our findings with regard to assisted reproductive techniques has to be made by taking into consideration intra-species differences between the mouse model and humans. Also, the sole effect of the cumulus cells' contribution could not be adequately addressed by comparing IVM COC and IVM DO, because the IVM DO were matured without gonadotropin supplementation. WIDER IMPLICATIONS OF THE FINDINGS: Our findings confirmed the inferiority of standard IVM technology compared with the in vivo approach. It also pointed at compromised biological processes employed in the critical translational regulation of in vitro matured MII oocytes and pronuclear zygotes. By highlighting the importance of proper translational regulation during in vitro oocyte maturation, this study should prompt further clinical investigations in the context of translation. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the Czech Grant Agency (22-27301S), Charles University Grant Agency (372621), Ministry of Education, Youth and Sports (EXCELLENCE CZ.02.1.01/0.0/0.0/15_003/0000460 OP RDE), and Institutional Research Concept RVO67985904. No competing interest is declared.

Reproductive outcomes of dual trigger therapy with GnRH agonist and hCG versus hCG trigger in women with diminished ovarian reserve: a retrospective study.

BACKGROUND: Diminished ovarian reserve (DOR) is one of the obstacles affecting the reproductive outcomes of patients receiving assisted reproductive therapy. The purpose of this study was to investigate whether dual trigger, including gonadotropin-releasing hormone agonist (GnRHa) and human chorionic gonadotropin (hCG), can improve pregnancy outcomes in patients with DOR undergoing in vitro fertilization (IVF) cycles using mild stimulation protocols. METHODS: A total of 734 patients with DOR were included in this retrospective study. Patients were divided into a recombinant hCG trigger group and a dual trigger group (hCG combined with GnRHa) according to the different trigger drugs used. The main outcome measures included the number of oocytes retrieved, the fertilization rate, the number of transferable embryos, the implantation rate, the clinical pregnancy rate, the miscarriage rate, the live birth rate (LBR), and the cumulative live birth rate (CLBR). Generalized linear model and logistic regression analyses were performed for confounding factors. RESULTS: There were 337 cycles with a single hCG trigger and 397 cycles with dual trigger. The dual trigger group demonstrated significantly higher numbers of retrieved oocytes [3.60 vs. 2.39, adjusted ß = 0.538 (0.221-0.855)], fertilized oocytes [2.55 vs. 1.94, adjusted ß = 0.277 (0.031-0.523)] and transferable embryos [1.22 vs. 0.95, adjusted ß = 0.162 (-0.005-0.329)] than did the hCG trigger group, whereas no significant difference in the fertilization rate was observed between the two groups. Moreover, the embryo transfer cancellation rate (35.5% vs. 43.9%) was obviously lower in the dual trigger group. Among the fresh embryo transfer cycles, the implantation rate, clinical pregnancy rate, miscarriage rate and live birth rate were similar between the two groups. After controlling for potential confounding variables, the trigger method was identified as an independent factor affecting the number of oocytes retrieved but had no significant impact on the CLBR. CONCLUSIONS: Dual triggering of final oocyte maturation with hCG combined with GnRHa can significantly increase the number of oocytes retrieved in patients with DOR but has no improvement effect on the implantation rate, clinical pregnancy rate or LBR of fresh cycles or on the CLBR.

Enhancing endometrial receptivity: the roles of human chorionic gonadotropin in autophagy and apoptosis regulation in endometrial stromal cells.

Inadequate endometrial receptivity often results in embryo implantation failure and miscarriage. Human chorionic gonadotropin (hCG) is a key signaling molecule secreted during early embryonic development, which regulates embryonic maternal interface signaling and promotes embryo implantation. This study aimed to examine the impact of hCG on endometrial receptivity and its underlying mechanisms. An exploratory study was designed, and endometrial samples were obtained from women diagnosed with simple tubal infertility or male factor infertile (n = 12) and recurrent implantation failure (RIF, n = 10). Using reverse transcription-quantitative PCR and western blotting, luteinizing hormone (LH)/hCG receptor (LHCGR) levels and autophagy were detected in the endometrial tissues. Subsequently, primary endometrial stromal cells (ESCs) were isolated from these control groups and treated with hCG to examine the presence of LHCGR and markers of endometrial receptivity (HOXA10, ITGB3, FOXO1, LIF, and L-selectin ligand) and autophagy-related factors (Beclin1, LC3, and P62). The findings revealed that the expressions of receptivity factors, LHCGR, and LC3 were reduced in the endometrial tissues of women with RIF compared with the control group, whereas the expression of P62 was elevated. The administration of hCG to ESCs specifically activated LHCGR, stimulating an increase in the endometrial production of HOXA10, ITGB3, FOXO1, LIF and L-selectin ligands. Furthermore, when ESCs were exposed to 0.1 IU/mL hCG for 72 h, the autophagy factors Beclin1 and LC3 increased within the cells and P62 decreased. Moreover, the apoptotic factor Bax increased and Bcl-2 declined. However, when small interfering RNA was used to knock down LHCGR, hCG was less capable of controlling endometrial receptivity and autophagy molecules in ESCs. In addition, hCG stimulation enhanced the phosphorylation of ERK1/2 and mTOR proteins. These results suggest that women with RIF exhibit lower levels of LHCGR and compromised autophagy function in their endometrial tissues. Thus, hCG/LHCGR could potentially improve endometrial receptivity by modulating autophagy and apoptosis.

Gonadotropin elevation is ootoxic to ovulatory oocytes and inhibits oocyte maturation, and activin decoy receptor ActRIIB:Fc therapeutically restores maturation.

BACKGROUND: Elevated FSH often occurs in women of advanced maternal age (AMA, age ≥ 35) and in infertility patients undergoing controlled ovarian stimulation (COS). There is controversy on whether high endogenous FSH contributes to infertility and whether high exogenous FSH adversely impacts patient pregnancy rates. METHODS: The senescence-accelerated mouse-prone-8 (SAMP8) model of female reproductive aging was employed to assess the separate impacts of age and high FSH activity on the percentages (%) of viable and mature ovulated oocytes recovered after gonadotropin treatment. Young and midlife mice were treated with the FSH analog equine chorionic gonadotropin (eCG) to model both endogenous FSH elevation and exogenous FSH elevation. Previously we showed the activin inhibitor ActRIIB:Fc increases oocyte quality by preventing chromosome and spindle misalignments. Therefore, ActRIIB:Fc treatment was performed in an effort to increase % oocyte viability and % oocyte maturation. RESULTS: The high FSH activity of eCG is ootoxic to ovulatory oocytes, with greater decreases in % viable oocytes in midlife than young mice. High FSH activity of eCG potently inhibits oocyte maturation, decreasing the % of mature oocytes to similar degrees in young and midlife mice. ActRIIB:Fc treatment does not prevent eCG ootoxicity, but it restores most oocyte maturation impeded by eCG. CONCLUSIONS: FSH ootoxicity to ovulatory oocytes and FSH maturation inhibition pose a paradox given the well-known pro-growth and pro-maturation activities of FSH in the earlier stages of oocyte growth. We propose the FOOT Hypothesis ("FSH OoToxicity Hypothesis), that FSH ootoxicity to ovulatory oocytes comprises a new driver of infertility and low pregnancy success rates in DOR women attempting spontaneous pregnancy and in COS/IUI patients, especially AMA women. We speculate that endogenous FSH elevation also contributes to reduced fecundity in these DOR and COS/IUI patients. Restoration of oocyte maturation by ActRIB:Fc suggests that activin suppresses oocyte maturation in vivo. This contrasts with prior studies showing activin A promotes oocyte maturation in vitro. Improved oocyte maturation with agents that decrease endogenous activin activity with high specificity may have therapeutic benefit for COS/IVF patients, COS/IUI patients, and DOR patients attempting spontaneous pregnancies.

Pulsatile Gonadotropin-Releasing Hormone Therapy Is Associated With Better Spermatogenic Outcomes than Gonadotropin Therapy in Patients With Pituitary Stalk Interruption Syndrome.

OBJECTIVE: To compare the effects of combined gonadotropin and pulsatile gonadotropin-releasing hormone (GnRH) therapy on spermatogenesis in patients with pituitary stalk interruption syndrome (PSIS). METHODS: Male patients with PSIS (N = 119) were retrospectively studied. Patients received pulsatile GnRH therapy (N = 59) were divided into response and poor-response groups based on luteinizing hormone (LH) levels after 1-month treatment with a cutoff value of 1 or 2 IU/L. Participants with gonadotropin therapy were divided into human menopausal gonadotropin (hMG)/human chorionic gonadotropin (hCG) group (N = 60), and patients with pulsatile GnRH therapy were classified into GnRH group (N = 28) with treatment duration ≥6 months. RESULTS: The overall success rates of spermatogenesis for hMG/hCG and GnRH therapy were 51.67% (31/60) vs 33.90% (20/59), respectively. GnRH group required a shorter period to induce spermatogenesis (8 vs 15 months, P = .019). hMG/hCG group had higher median total testosterone than GnRH group [2.16, interquartile range(IQR) 1.06-4.89 vs 1.31, IQR 0.21-2.26 ng/mL, P = .004]. GnRH therapy had a beneficial effect on spermatogenesis compared to hMG/hCG therapy (hazard ratio 1.97, 95% confidence interval 1.08-3.57, P = .026). In patients with pulsatile GnRH therapy, compared with the poor-response group, the response group had a higher successful spermatogenesis rate (5.00% vs 48.72%, P = .002) and higher median basal total testosterone (0.00, IQR 0.00-0.03 vs 0.04, IQR 0.00-0.16 ng/mL, P = .026) with LH = 1 IU/L as the cutoff value after 1-month pulsatile GnRH therapy. CONCLUSIONS: Pulsatile GnRH therapy was superior to hMG/hCG therapy for spermatogenesis in patients with PSIS. Earlier spermatogenesis and higher concentrations of sperm could be obtained in the GnRH group if patients received therapy over 6 months.

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.

Equine chorionic gonadotropin treatment and timed artificial insemination for dairy cow production under heat stress.

This study investigated the effects of timed artificial insemination (TAI) and equine chorionic gonadotropin (eCG) administration on lactating dairy cows under heat-stress conditions (average temperature-humidity index: 80). Timed artificial insemination was performed on the cows with (n = 57) or without (control, n = 41) supplementation with 500 IU of eCG at the day of PGF2α treatment using the CIDR-Ovsynch protocol. GnRH was administered, and a progesterone device (CIDR) was inserted on Day -10 of the treatment protocol. The CIDR was removed on Day -3, and the cows were treated with PGF2α. Two days later, a 2nd GnRH injection was administered. Subsequently, AI was performed on Day 0 (16-20 h after the 2nd GnRH injection), and pregnancy was diagnosed on Days 32 and 60. Plasma progesterone (P4) concentrations were measured after AI. Results showed that the eCG group had a higher pregnancy per AI (P/AI) than the control group (43.9 vs. 12.2%, P = 0.002), which was also accompanied by elevated P4 levels. Four cows in the eCG group had multiple calves, representing 7.0 and 16.0% of the group and pregnant cows, respectively. In conclusion, 500 IU of eCG combined with CIDR-Ovsynch in lactating dairy cows under severe heat stress conditions successfully improved fertility. However, the protocol may have a slight risk of multiple births.

Luteinizing hormone supplementation with human menopausal gonadotropin versus low dose human chorionic gonadotropin during ovarian stimulation does not affect live birth rates after fresh and frozen embryo transfer.

AIM: Luteinizing hormone (LH) plays an important role in ovarian follicle maturation. Human menopausal gonadotropin (hMG) or low dose human chorionic gonadotropin (hCG) can provide LH supplementation during in vitro fertilization (IVF) ovarian stimulation, though studies directly comparing their impact on IVF outcomes are limited. The aim of the study was to determine whether LH supplementation with hMG versus low dose hCG during IVF stimulation affects live birth rate. METHODS: Fresh and frozen embryo transfers (ET) from 2017 to 2021 after standard long or antagonist protocols supplemented with hMG (75-250 IU) or low dose hCG (50-100 IU) during stimulation cycles in our academic center were included. Statistical analysis was performed with T-tests, Mann-Whitney U tests, Chi-square, and multiple linear and logistic regression. RESULTS: Four hundred and sixty eight unique stimulation cycles resulting in 213 fresh and 412 frozen embryo transfers were analyzed. There was a lower mature oocyte yield (10.9 vs. 11.8, p = 0.044) but similar high-quality blastocyst yield (3.6 vs. 3.9, p = 0.11) for hMG vs low dose hCG. Live birth rates per transfer were comparable for fresh (42% vs. 49%, p = 0.24) and frozen (46% vs. 53%, p = 0.45) embryo transfers. Multiple logistic regressions showed no association between supplemental gonadotropin and live birth for both fresh and frozen embryo transfers. CONCLUSION: Fresh and frozen IVF-ET pregnancy outcomes were comparable after hMG versus low dose hCG supplementation, suggesting flexibility in supplemental LH dosing regimens that may address patient or physician preference or cost concerns.

Rescue in vitro maturation using ovarian support cells of human oocytes from conventional stimulation cycles yields oocytes with improved nuclear maturation and transcriptomic resemblance to in vivo matured oocytes.

PURPOSE: Determine if the gene expression profiles of ovarian support cells (OSCs) and cumulus-free oocytes are bidirectionally influenced by co-culture during in vitro maturation (IVM). METHODS: Fertility patients aged 25 to 45 years old undergoing conventional ovarian stimulation donated denuded immature oocytes for research. Oocytes were randomly allocated to either OSC-IVM culture (intervention) or Media-IVM culture (control) for 24-28 h. The OSC-IVM culture condition was composed of 100,000 OSCs in suspension culture with human chorionic gonadotropin (hCG), recombinant follicle stimulating hormone (rFSH), androstenedione, and doxycycline supplementation. The Media-IVM control lacked OSCs and contained the same supplementation. A limited set of in vivo matured MII oocytes were donated for comparative evaluation. Endpoints consisted of MII formation rate, morphological and spindle quality assessment, and gene expression analysis compared to in vitro and in vivo controls. RESULTS: OSC-IVM resulted in a statistically significant improvement in MII formation rate compared to the Media-IVM control, with no apparent effect on morphology or spindle assembly. OSC-IVM MII oocytes displayed a closer transcriptomic maturity signature to IVF-MII controls than Media-IVM control MII oocytes. The gene expression profile of OSCs was modulated in the presence of oocytes, displaying culture- and time-dependent differential gene expression during IVM. CONCLUSION: The OSC-IVM platform is a novel tool for rescue maturation of human oocytes, yielding oocytes with improved nuclear maturation and a closer transcriptomic resemblance to in vivo matured oocytes, indicating a potential enhancement in oocyte cytoplasmic maturation. These improvements on oocyte quality after OSC-IVM are possibly occurring through bidirectional crosstalk of cumulus-free oocytes and ovarian support cells.

Effect of GnRH agonist trigger with or without low-dose hCG on reproductive outcomes for PCOS women with freeze-all strategy: a propensity score matching study.

PURPOSE: This study aimed to compare the effect of gonadotropin-releasing hormone agonist (GnRHa) trigger alone versus dual trigger comprising GnRHa and low-dose human chorionic gonadotropin (hCG) on reproductive outcomes in patients with polycystic ovary syndrome (PCOS) who received the freeze-all strategy. METHODS: A total of 615 cycles were included in this retrospective cohort study. Propensity score matching (PSM) was performed to control potential confounding factors between GnRHa-trigger group (0.2 mg GnRHa) and dual-trigger group (0.2 mg GnRHa plus 1000/2000 IU hCG) in a 1:1 ratio. Multivariate logistic regression was applied to estimate the association between trigger methods and reproductive outcomes. RESULTS: After PSM, patients with dual trigger (n = 176) had more oocytes retrieved, mature oocytes, and 2PN embryos compared to that with GnRHa trigger alone. However, the oocytes maturation rate, normal fertilization rate, and frozen embryos between the two groups were not statistically different. The incidence of ovarian hyperstimulation syndrome (OHSS) (14.8% vs. 2.8%, P < 0.001) and moderate/severe OHSS (11.4% vs. 1.7%, P < 0.001) were significantly higher in dual-trigger group than in GnRHa-alone group. Logistic regression analysis showed the adjusted odds ratio of dual trigger was 5.971 (95% confidence interval 2.201-16.198, P < 0.001) for OHSS. The pregnancy and single neonatal outcomes were comparable between the two groups (P > 0.05). CONCLUSION: For PCOS women with freeze-all strategy, GnRHa trigger alone decreased the risk of OHSS without damaging oocyte maturation and achieved satisfactory pregnancy outcomes.

The effect of human menopausal gonadotropin and equine chorionic gonadotropin on the reproductive performance of treated ewes with short-term progesterone injections and sponge during the non-breeding season.

This study investigated the effect of human menopausal gonadotropin (hMG) on reproductive efficiency of synchronized ewes with the sponge and progesterone (P4) injection-based protocols. In study 1, anoestrous ewes (n = 120) were used. Sixty ewes were treated with sponge (S) for 12 days. The injection of eCG (SeCG group, n = 30) or hMG (ShMG, n = 30) was given at the time of sponge removal. Thirty ewes received IM injection of P4, three times every 48 h and the injection of hMG was given 24 h after the third P4 injection (3PhMG group, n = 30), and 30 ewes were used as control group. Pregnancy was diagnosed on day 50 after the release of ram. In study 2, 60 ewes were randomly divided into two equal groups. In the treated group with antibiotics (n = 30), before inserting, the sponges were impregnated with the antibiotic penicillin G sodium (5,000,000 IU) and in the control group (n = 30), there was no added antibiotics. Before inserting and after removing sponges, a vaginal cytology sample was taken with a sterile cotton swab. The number of neutrophils in each sample was counted and analysed. The rate of oestrus and total pregnancy was greater in SeCG (96.7, 93.3%), ShMG (82.8, 93.1%) and 3PhMG (67.9, 89.3%) groups compared with the control group (13.8, 41.4%) (p < .05). No significant difference was found in single, twin and total lambing and pregnancy rates after injection of eCG and hMG during the non-breeding season (p > .05). A higher percentage of control ewes had the vaginal smear with neutrophils more than 50% (96.7% vs. 76.7%; p < .05). In conclusion, a single dose of hMG can induce fertile oestrus in synchronized ewes with P4 administered by either injection or intravaginally. Purulent discharge and percentage of neutrophils were significantly reduced in the synchronized ewes by the impregnated sponges with the antibiotic penicillin.

Optimising Laparoscopic Artificial Insemination in Lohi Sheep: Effects of Timing, Sperm Concentration and Body Condition on Fertility Outcome in Subtropical Climates.

The objective of this study was to determine the optimal timing, sperm concentration, and body condition score (BCS) for laparoscopic artificial insemination (LAI) in the subtropical Lohi sheep breed. In Experiment 1, Lohi ewes (n = 80) were synchronised through progestin-sponges (day 0-day 11), administering PGF2α (d-cloprostenol 75 µg/mL; i.m. @ 75 µg /ewe) on day 09 and eCG (i.m. @ 300 IU/ewe) on day 11. Ewes were divided equally into four groups (n = 20 each) and then LAI was performed at 48 (T48), 60 (T60), 72 (T72) and 96 (T96) hours post-sponge removal using 200 million sperm/insemination. In Experiment 2, ewes (n = 81) were synchronised as in Experiment I. Following synchronisation ewes were divided into four groups, each subjected to LAI employing varying sperm concentrations: 10 (S10; n = 21), 20 (S20; n = 20), 50 (S50; n = 20), or 100 (S100; n = 20) million per insemination. Inseminations were performed within a time window of 48-60 h post-sponge removal, based on the findings from Experiment 1. In both experiments ewes were categorised according to BCS, that is, medium 3 and high > 3. Results of Experiment 1 revealed a quadratic response that ewes inseminated at 60-h post-sponge removal exhibited significantly higher pregnancy and twinning rates (p = 0.02). The results of Experiment 2 revealed that sperm concentration had a quadratic effect, 50 million sperm per insemination resulted in maximal pregnancy rates (p = 0.01). Additionally, ewes with medium BCS (≤ 3) had higher pregnancy rates than high BCS (> 3) ewes. In conclusion, ewes with medium BCS (≤ 3) are ideal candidate for LAI and can be inseminated at 60 h with minimal sperm 50 × 106/dose post-sponge removal to achieve a maximum pregnancy rate.

Morphometric Evaluation of the Ampullae of the Oviducts in Prepubertal Gilts Treated With Chorionic Gonadotropins.

This study assessed morphometric traits of the ampulla of the oviducts in prepubertal gilts treated with chorionic gonadotropins. With the day of slaughter as D0, gilts were assigned to four treatments (n = 8 each): control (untreated), eCG (200 IU eCG on D3), eCG+hCG (1200 IU eCG on D6 plus 500 IU hCG on D3), and eCG+hCG+AI (the previous treatment plus artificial insemination on D1). Blood and ampullae samples were collected at slaughter. Serum progesterone concentrations were higher for gilts treated with hCG than for those in the eCG and control treatments (p < 0.001), but estradiol concentrations did not differ (p > 0.05). The epithelium, muscle and lumen areas and the inner and larger ampullae diameters did not differ across treatments (p > 0.05). Therefore, treatment with chorionic gonadotropins did not alter the ampullae morphometry of prepubertal gilts.

Associations among the largest follicle, preovulatory estradiol concentrations, and predominant vaginal epithelial cells at the completion of hormonal ovarian stimulation for fixed-time artificial insemination in goats.

The objective of the present study was to investigate the association among the largest follicle (LF), preovulatory estradiol (E2), and predominant vaginal epithelial cell at the completion of hormonal ovarian stimulation for fixed-time artificial insemination (FTAI) in goats. Thirty-seven crossbred Boer does received gonadotropin-releasing hormone (GnRH) and intravaginal progesterone (P4)-releasing devices (day 0). On day 5, P4 devices were removed and does received prostaglandin F2α and equine chorionic gonadotrophin. On day 7, does received GnRH, and FTAI was undertaken. On day 7, does were divided into three groups, i.e. small-sized (3-3.9 mm; n = 5), medium-sized (4-4.9 mm; n = 8), and large-sized (≥5 mm; n = 24) according to the diameter of the ovarian LF; follicular characteristics (number and diameter) were identified, and blood samples and vaginal smears were collected. The average diameters of total antral follicles and LF and the percentage of superficial cell were greatest in large-sized LF does (p < .01). The average diameters of total antral follicle (r = .68) and LF (r = .71), number of preovulatory follicle (r = .58), and plasma E2 concentrations (r = .61) were positively correlated with the percentage of superficial cells (p < .01). The likelihood of a pregnancy outcome after the FTAI increased by 13.71 times in does with a greater average diameter of antral follicle, 14.18 times with emergence of a large preovulatory follicle, and 36.83 times with a higher percentage of vaginal superficial cells (p < .01). It was concluded that there is a relationship between the cell types of the vaginal epithelium, the diameters of the largest ovarian follicles, and the concentration of E2 in goats subjected to FTAI protocols.

Stable Production of a Recombinant Single-Chain Eel Follicle-Stimulating Hormone Analog in CHO DG44 Cells.

This study aimed to produce single-chain recombinant Anguillid eel follicle-stimulating hormone (rec-eel FSH) analogs with high activity in Cricetulus griseus ovary DG44 (CHO DG44) cells. We recently reported that an O-linked glycosylated carboxyl-terminal peptide (CTP) of the equine chorionic gonadotropin (eCG) ß-subunit contributes to high activity and time-dependent secretion in mammalian cells. We constructed a mutant (FSH-M), in which a linker including the eCG ß-subunit CTP region (amino acids 115-149) was inserted between the ß-subunit and α-subunit of wild-type single-chain eel FSH (FSH-wt). Plasmids containing eel FSH-wt and eel FSH-M were transfected into CHO DG44 cells, and single cells expressing each protein were isolated from 10 and 7 clones. Secretion increased gradually during the cultivation period and peaked at 4000-5000 ng/mL on day 9. The molecular weight of eel FSH-wt was 34-40 kDa, whereas that of eel FSH-M increased substantially, with two bands at 39-46 kDa. Treatment with PNGase F to remove the N glycosylation sites decreased the molecular weight remarkably to approximately 8 kDa. The EC50 value and maximal responsiveness of eel FSH-M were approximately 1.23- and 1.06-fold higher than those of eel FSH-wt, indicating that the mutant showed slightly higher biological activity. Phosphorylated extracellular-regulated kinase (pERK1/2) activation exhibited a sharp peak at 5 min, followed by a rapid decline. These findings indicate that the new rec-eel FSH molecule with the eCG ß-subunit CTP linker shows potent activity and could be produced in massive quantities using the stable CHO DG44 cell system.

The Effects of Human Chorionic Gonadotropin and Gonadotropin-Releasing Hormone Receptor Antagonist on Testicular Steroidogenesis in Normal and Obese Rats.

We studied the effect of a high-fat, high-carbohydrate diet (HFHCD) on basal testosterone levels in the blood and testosterone, its precursors, and expression of steroidogenic genes in the testes of rats treated with human chorionic gonadotropin (hCG, 10 IU/rat, subcutaneously, once), gonadotropin-releasing hormone receptor antagonist cetrorelix (75 µg/kg, subcutaneously, 3 days), and their combination. In HFHCD rats, no obvious signs of androgen deficiency were observed and the response of the testes to hCG stimulation was preserved. Unlike control rats (normal diet), the expression of the luteinizing hormone receptor gene in these rats did not change in response to hCG stimulation and cetrorelix administration; they also showed a paradoxical, more pronounced response to hCG administration under conditions of suppression of the gonadotropin secretion by cetrorelix. This suggests that the etiology and pathogenesis of obesity may have different effects on the hormonal status of the male reproductive system.

Divergent roles of sirtuin 1 in human granulosa-lutein cells: similarities to human chorionic gonadotropin.

Sirtuin 1 (SIRT1) is a nicotinamide adenine dinucleotide-dependent deacetylase that modifies gene expression through histone deacetylation. It also deacetylates nonhistone substrates, e.g., tumor suppressor p53, NOS3, HIF1A, NFKB, FOXO3a, PGC-1α, and PPARγ. Consequently, it regulates a wide range of physiological functions including cell cycle control, energy expenditure, oxidative stress response, apoptosis, and aging. SIRT1 is expressed in ovarian granulosa cells (GCs) of various species including humans at different stages of the reproductive cycle. The importance of SIRT1 in female reproduction is supported by the findings that SIRT1-knockout mice exhibit defects in reproductive tissue development. These mice were found to have a thin-walled uterus, small ovaries, with follicles present but no corpora lutea. This review aims to provide state-of-the-art information on SIRT1's mode of action and its roles in human granulosa-lutein cells and GCs from other species where data are available. It also discusses the overlapping actions of SIRT1 and human chorionic gonadotropin on the production of critical GC-borne factors.

Exposure to a phthalate mixture disrupts ovulatory progesterone receptor signaling in human granulosa cells in vitro†.

Exposure to phthalates disrupts ovarian function. However, limited studies have investigated the effects of phthalate mixtures on ovulation, especially in women. Human granulosa cells were used to test the hypothesis that exposure to a phthalate mixture (PHTmix) disrupts progesterone (P4)/progesterone receptor (PGR) signaling, which is a crucial pathway for ovulation. In addition, progestin and cyclic adenosine 3', 5'-monophosphate (cAMP) supplementation were tested as methods to circumvent phthalate toxicity. Granulosa cells from women undergoing in vitro fertilization were acclimated in culture to regain responsiveness to human chorionic gonadotropin (hCG; clinical luteinizing hormone analogue). Granulosa cells were treated with or without hCG, and with or without PHTmix (1-500 µg/ml; dimethylsulfoxide = vehicle control) for 0.5-36 h. In the supplementation experiments, cells were treated with or without R5020 (stable progestin), and with or without 8-Br-cAMP (stable cAMP analogue). Exposure to hCG + PHTmix decreased P4 levels and mRNA levels of steroidogenic factors when compared to hCG. This was accompanied by decreased mRNA levels of PGR and downstream P4/PGR ovulatory mediators (ADAM metallopeptidase with thrombospondin type 1 motif 1 (ADAMTS1), C-X-C motif chemokine receptor 4 (CXCR4), pentraxin 3 (PTX3), and regulator of G protein signaling 2 (RGS2)) in the hCG + PHTmix groups compared to hCG. Exposure to hCG + PHTmix 500 µg/ml decreased cAMP levels and protein kinase A activity compared to hCG. Supplementation with progestin in the hCG + PHTmix 500 µg/ml group did not rescue toxicity, while supplementation with cAMP restored PGR levels and downstream P4/PGR mediator levels to hCG levels. These findings suggest that phthalate mixture exposure inhibits P4/PGR signaling in human granulosa cells via decreased steroidogenesis, cAMP levels, and protein kinase A activity. Restored P4/PGR signaling with cAMP supplementation provides a potential cellular target for intervention of phthalate-induced ovulatory dysfunction in women.

Neurotensin expression, regulation, and function during the ovulatory period in the mouse ovary†.

The luteinizing hormone (LH) surge induces paracrine mediators within the ovarian follicle that promote ovulation. The present study explores neurotensin (NTS), a neuropeptide, as a potential ovulatory mediator in the mouse ovary. Ovaries and granulosa cells (GCs) were collected from immature 23-day-old pregnant mare serum gonadotropin primed mice before (0 h) and after administration of human chorionic gonadotropin (hCG; an LH analog) across the periovulatory period (4, 8, 12, and 24 h). In response to hCG, Nts expression rapidly increased 250-fold at 4 h, remained elevated until 8 h, and decreased until 24 h. Expression of Nts receptors for Ntsr1 remained unchanged across the periovulatory period, Ntsr2 was undetectable, whereas Sort1 expression (also called Ntsr3) gradually decreased in both the ovary and GCs after hCG administration. To better understand Nts regulation, inhibitors of the LH/CG signaling pathways were utilized. Our data revealed that hCG regulated Nts expression through the protein kinase A (PKA) and p38 mitogen-activated protein kinase (p38MAPK) signaling pathways. Additionally, epidermal-like-growth factor (EGF) receptor signaling also mediated Nts induction in GCs. To elucidate the role of NTS in the ovulatory process, we used a Nts silencing approach (si-Nts) followed by RNA-sequencing (RNA-seq). RNA-seq analysis of GCs collected after hCG with or without si-Nts identified and qPCR confirmed Ell2, Rsad2, Vps37a, and Smtnl2 as genes downstream of Nts. In summary, these findings demonstrate that hCG induces Nts and that Nts expression is mediated by PKA, p38MAPK, and EGF receptor signaling pathways. Additionally, NTS regulates several novel genes that could potentially impact the ovulatory process.