The Role of Traditional Chinese Formula Ding-Kun Pill (DKP) in Expected Poor Ovarian Response Women (POSEIDON Group 4) Undergoing In Vitro Fertilization-Embryo Transfer: A Multicenter, Randomized, Double-Blind, Placebo-Controlled Trial.

Objective: The primary objective of the study was to assess traditional Chinese formula DKP supplementation in terms of efficacy and safety on reproductive outcomes of expected poor ovarian responder (POR, POSEIDON Group 4) undergoing in vitro fertilization-embryo transfer (IVF-ET). Design Setting and Participants: Women eligible for IVF-ET were invited to participate in this randomized, double-blind, placebo-controlled, superiority trial at academic fertility centers of ten public hospitals in Chinese Mainland. A total of 462 patients (35-44 years) equally divided between DKP and placebo groups with antral follicle count (AFC) <5 or anti-müllerian hormone (AMH) <1.2 ng/ml were randomized. Interventions: All participants were given DKP or 7 g placebo twice daily on the previous menstrual cycle day 5 until oocyte retrieval, which took approximately 5 to 6 weeks. Main Outcome Measure: The primary outcome was the ongoing pregnancy defined as more than 20 gestational weeks of an intrauterine living fetus confirmed by pelvic ultrasonography. Results: Demographic characteristics were equally distributed between the study populations. Intention-to-treat (ITT) analysis revealed that ongoing pregnancy rate (OPR) was not significantly different between DKP and placebo groups [26.4% (61/231) versus 24.2% (56/231); relative risk (RR) 1.09, 95% confidence interval (CI) 0.80 to 1.49, P = 0.593]. No significant differences between groups were observed for the secondary outcomes. The additional per protocol (PP) analysis was in line with ITT results: OPR in DKP group was 27.2% (61/224) versus 24.1% (55/228) in placebo group [RR 1.13, 95%CI (0.82 to 1.55), P = 0.449]. After subgroup analysis the findings concluded that POR population of 35-37 years had a significantly higher OPR after 5-6 weeks of oral DKP (41.8%, 33/79) versus placebo (25.4%, 18/71) [RR 1.65, 95% CI (1.02 to 2.65), P = 0.034, P for interaction = 0.028]. Conclusion: This well-designed randomized controlled trial (RCT) offers new high-quality evidence to supplement existing retrospective literature concerning DKP performance in expected PORs. DKP could be recommended as a safe and natural remedy for expected PORs (aged 35-37 years) who fulfill the POSEIDON group 4 criteria. However, additional interventional clinical studies are undoubtedly required to be conducted in the future to validate this hypothesis. Clinical Trial Registration: www.chictr.org.cn, identifier ChiCTR1900026614.

Use of a male antioxidant nutraceutical is associated with superior live birth rates during IVF treatment.

Oxidative stress is prevalent among infertile men and is a significant cause of sperm DNA damage. Since sperm DNA damage may reduce embryo quality and increase miscarriage rates, it is possible that untreated sperm oxidative stress may impair in vitro fertilization (IVF) live birth rates. Given that the antioxidant Menevit is reported to reduce sperm DNA damage, it was hypothesized that men's consumption of this supplement may alter IVF outcomes. Therefore, a retrospective cohort study was conducted analyzing outcomes for couples undergoing their first fresh embryo transfer. Men were classified as controls if they were taking no supplements, health conscious controls if taking "general health" supplements, or Menevit users. Men with karyotype abnormalities, or cycles using donated, frozen and surgically extracted sperm were excluded. Among the final study cohort of 657 men, live birth rates were significantly higher in Menevit users than controls (multivariate adjusted odds ratio [OR]: 1.57, 95% confidence interval [CI]: 1.01-2.45, P= 0.046), but not between controls taking no supplements and those using general health supplements, thereby suggesting that potential health conscious behavior in supplement users is unlikely responsible for the superior outcomes in Menevit users. Interestingly, in a post hoc sensitivity analysis, live birth rates among Menevit users were statistically superior to controls for lean men (OR: 2.73, 95% CI: 1.18-6.28; P= 0.019), not their overweight/obese counterparts (OR: 1.29, 95% CI: 0.75-2.22, P = 0.37). The results of this large cohort study therefore support a positive association between men's use of the Menevit antioxidant during IVF treatment and live birth rates, especially in lean individuals.

The effect of myo-inositol, vitamin D3 and melatonin on the oocyte quality and pregnancy in in vitro fertilization: a randomized prospective controlled trial.

OBJECTIVE: To evaluate the efficacy of a treatment with Myo-inositol (MI) plus melatonin and vitamin D3 in women underwent intra cytoplasmatic sperm injection (ICSI). PATIENTS AND METHODS: 100 women undergoing ICSI procedure were enrolled and randomly divided 1:1 in two groups. The study group was treated with 2 g MI, 50 mg Alpha-Lactalbumin (alpha-LA) and 200 µg folic acid in powder every morning for 6 months (3 months before oocyte pick up and 3 months after ICSI); the same patients underwent treatment with 600 mg MI, 1 mg melatonin plus 200 µg folic acid during the evening for 3 months before oocyte pick up; subsequently the pick up these patients were treated with 600 mg MI, 200 µg folic acid, 1 mg melatonin, 50 µg vitamin D3 as cholecalciferol until the 12th week of gestation. The control group was treated with 200 µg folic acid twice a day. Clinical pregnancy rate was evaluated as primary outcome, followed by blastocyst and oocyte quality, as well as gestational period as secondary outcomes. RESULTS: Treatment significantly improved blastocyst and oocyte quality in the study group, achieving the 42% of clinical pregnancies vs. 24% in the control group, even though the course of pregnancy did not significantly differ between the groups. However, the mean gestational period was shorter in the control group. CONCLUSIONS: The supplementation of MI in combination with melatonin in the first 3 months before oocyte pick up and with vitamin D3 in the further 3 months could represent an innovative support for all those women undergoing ICSI.

Protective effect of vitamin C and lycopene on the in vitro fertilization capacity of sex-sorted bull sperm by inhibiting the oxidative stress.

The fertilization capacity of sex-sorted sperms is seriously decreased, which inhibits its wide application. However, little information is still available about the effect of vitamin C (VC) and lycopene (Lyc) on the fertilization capacity of sex-sorted bull sperm. In this study, the washing medium and fertilization medium of sex-sorted sperm from three bull individuals were supplemented with different concentrations of VC (0, 1 × 10-3 , 1 × 10-4 , 1 × 10-5 , 1 × 10-6  M) or Lyc (0, 1 × 10-4 , 1 × 10-5 , 1 × 10-6 , 1 × 10-7 ). After washing twice and incubation for 1.5 hr, the malondialdehyde (MDA) level, phosphatidylserine (PS) translocation, membrane potential (Δψm) and IVF (in vitro fertilization) ability of sex-sorted sperm were investigated. For the sex-sorted sperm of bulls A, B and C, 1 × 10-3  M VC or 1 × 10-4  M Lyc treatment significantly decreased their MDA levels and PS translocation and increased their Δψm levels and cleavage rates after IVF. When blastocysts were concerned, 1 × 10-4  M Lyc significantly improved the blastocyst rates and their IFN-tau expression of bulls A and C. In conclusion, supplementation of 1 × 10-3  M VC or 1 × 10-4  M Lyc in washing and fertilization medium contributed greatly to improving the fertilization capacity of sex-sorted bull sperm during IVF procedure.

Does Dehydroepiandrosterone (DHEA) improve in vitro fertilisation (IVF) outcomes in poor responders?

BACKGROUND: In reproductive medicine poor ovarian response (POR) among women undergoing in vitro fertilisation (IVF) is of great concern. Meta-analysis showed that Dehydroepiandrosterone (DHEA) administration resulted in a significant increase in the number of oocytes retrieved in women with POR. The aim of this study was to assess the effectiveness of DHEA supplementation on IVF outcomes among poor responders undergoing IVF. METHODS: Sixteen patients who were diagnosed with POR scheduled to undergo their second cycle of Intracytoplasmic sperm injection (ICSI)/embryo transfer cycle were enrolled. All enrolled patients had earlier undergone their first ICSI/embryo transfer cycle at least four months prior to this study. All subjects were given DHEA supplementation of 25mg three times daily for at least three months prior to their second ICSI/embryo transfer cycle. Statistical analysis of various ovarian response and ICSI outcomes parameter were compared pre and post DHEA. RESULTS: Sixteen women with the mean age of 35 years were enrolled in the study. The comparative analysis of results showed a significant increase in the number of good quality of embryos obtained (p<0.05). After the treatment with DHEA, there was an improvement in the number of oocytes retrieved, Metaphase II (MII) oocyte (mature) oocytes obtained, fertilised and transferrable embryos and the pregnancy rate. There was no significant effect of DHEA treatment on the number of days of stimulation and cumulative dose of gonadotrophins used. CONCLUSION: Our results is able to show that DHEA supplementation may help to enhance IVF-ICSI outcomes in women with POR especially in those age 35 years and below.

Supplementation of biotin to sperm preparation medium enhances fertilizing ability of spermatozoa and improves preimplantation embryo development.

PURPOSE: Motility of spermatozoa helps not only in planning the type of infertility treatment but also directly reflects the success rate in assisted reproductive technology (ART). Previously, biotin, a water-soluble vitamin, has been shown to increase the motility and longevity of cryopreserved human spermatozoa. The present study was designed to understand the molecular basis of the beneficial effects of presence of biotin in sperm wash medium on early embryo development. METHODS: The effect biotin supplementation to sperm wash medium on the sperm parameters were assessed in swim-up fraction of normozoospermic and asthenozoospermic ejaculates collected from infertile men. Fertilization and early embryo development was studied using Swiss albino mice. RESULTS: Even though both biotin and pentoxifylline (PTX) enhanced the motility of spermatozoa from normozoospermic and asthenozoospermic samples, biotin group exhibited higher in vitro survival. Using mouse model, we observed that presence of biotin or PTX in sperm wash medium improved the fertilization rate and blastocyst rate compared to control. Blastocysts from these groups had significantly higher total cell number (P < 0.01) and lower apoptotic index. In silico target prediction revealed that GTPase HRas (HRas), tyrosine-protein phosphatase nonreceptor type 1 (PTP1B), and glucokinase are the probable targets for biotin. Solution-state Nuclear Magnetic Resonance (NMR) studies confirmed that biotin interacts both with human HRas and PTP1B. CONCLUSION: Our results indicate that presence of biotin in sperm wash medium can improve the fertilization potential and preimplantation embryo development and can be considered as a safe alternate to PTX.

Urea influences amino acid turnover in bovine cumulus-oocyte complexes, cumulus cells and denuded oocytes, and affects in vitro fertilization outcome.

High-protein diets often lead to an increase in urea concentration in follicular fluid of dairy cows, which may reduce oocyte competence. In the present study, maturation media were supplemented with urea (0, 20, 40 mg/dl), and amino acids (AAs) turnover was evaluated in the 24-h spent media of specimens (cell types), bovine cumulus-oocyte complexes (COCs), cumulus cells (CCs), or denuded oocytes (DOs). The main effects of urea and cell type, and their interaction were significant on the individual turnover (expect threonine, glycine, and tyrosine) and total turnover, depletion, and appearance of AAs. The results showed a high level of urea and DOs increased the depletion of all AAs and that of essential and non-AAs, respectively. Sensitivity analysis revealed the highest sensitivity of isoleucine, lysine, and tryptophan to urea, especially in DOs. Principal component analysis (PCA) evaluated the strong correlations between the turnover of: (1) glutamine, aspartic acid or glycine, and developmental competence and fertilization of COCs; (2) serine, isoleucine, valine or glutamic acid, and cleavage rate of DOs; and (3) serine, glutamine, aspartic acid or alanine, and CCs viability. In conclusion, urea significantly changed the turnover of AAs by COCs, CCs and DOs, and reduced the subsequent developmental competence of bovine oocytes.

Importance of oil overlay for production of porcine embryos in vitro.

Technologies to edit the zygote genome have revolutionized biomedical research not only for the creation of animal models for the study of human disease but also for the generation of functional human cells and tissues through interspecies blastocyst complementation technology. The pig is the ideal species for these purposes due to its great similarity in anatomy and physiology to humans. Emerging biotechnologies require the use of oocytes and/or embryos of good quality, which might be obtained using in vitro production (IVP) techniques. However, the current porcine embryo IVP systems are still suboptimal and result in low monospermic fertilization and blastocyst formation rates and poor embryo quality. During recent years, intensive investigations have been performed to evaluate the influence of specific compounds on gametes and embryos and to avoid the use of undefined supplements (serum and serum derivate) in the incubation media. However, little consideration has been given to the use of the mineral oil (MO) to overlay incubation droplets, which, albeit being a routine component of the IVP systems, is a totally undefined and thus problematic product for the safety of gametes and embryos. In this review, we provide an overview on the advantages and disadvantages of using MO to cover the incubation media. We also review one important concern in IVP laboratories: the use of oils containing undetected contamination. Finally, we discuss the effects of different types of oils on the in vitro embryo production outcomes and the transfer of compounds from oil into the culture media.

Antioxidants reduce oxidative stress in follicular fluid of aged women undergoing IVF.

BACKGROUND: The status characterized by the imbalance between pro-oxidants and antioxidants molecules, defined as oxidative stress, has been suggested to be involved in the pathogenesis of subfertility in females. This study aims to evaluate the impact of a complete micronutrients supplementation on oxidative stress levels in follicular microenvironment as well as on in vitro fertilization (IVF) outcome. METHODS: This preliminary study was conducted between January 2014 and July 2015 at the Siena University Hospital Infertility Clinic. Serum and follicular fluid were collected from infertile women aged > 39 years who underwent two in vitro fertilization cycles: in the first cycle they were treated with GnRH-antagonist protocol and gonadotropins for controlled ovarian hyperstimulation, whereas in the second cycle ovarian stimulation protocol was associated to micronutrients supplementation, starting three months earlier. Protein oxidation levels and total antioxidant capacity in serum and in follicular fluid were evaluated in IVF cycles with or without micronutrients supplementation. Differences in IVF outcome parameters were statistically evaluated. RESULTS: Two-dimensional electrophoresis analyses demonstrated that when patients assumed micronutrients before IVF cycles, follicular fluid and serum proteins were protected from oxidative damage. Comparable results were obtained when total antioxidant capacity was measured. Moreover, the mean number of good quality oocytes retrieved when patients received micronutrients supplementation was significantly increased. CONCLUSION: The additional treatment with micronutrients, starting three months before IVF cycles, protects the follicular microenvironment from oxidative stress, thus increasing the number of good quality oocytes recovered at the pick up.

Effects of aqueous crude extract of Echeveria gibbiflora on mouse sperm function.

UNLABELLED: The present study evaluates the possible antifertility effect of aqueous crude extract (OBACE) of Echeveria gibbiflora, a plant that belongs to the crassulaceae family, used in traditional Mexican medicine as a vaginal post coital rinse to prevent pregnancy and shown to have an immobilization/agglutination effect on sperm of different mammal species. We evaluated the effect of OBACE on functional parameters of mouse sperm, such as viability, capacitation, and acrosome reaction. In addition, due to the high concentrations of calcium bis-(hydrogen-1-malate) hexahydrate [Ca (C4H5O5)2•6H2O] present in this plant extract, we evaluated its effect on Ca(2+) influx in mouse sperm under capacitating conditions. Moreover, we determined the acute toxicity of OBACE and its in vivo effect in mouse sperm motility administering a single daily dose of 50 and 100 mg/kg during seven days, intraperitoneally. The sperm viability was not affected by the presence of different concentrations of OBACE, however, the capacitation and acrosome reaction suffered a significant decrease in a concentration-dependent manner, coinciding with the reduction of Ca(2+) influx. Furthermore, OBACE displayed an LD50 of 3,784.42 mg/kg and can be classified as a low toxic substance. Also, in vivo OBACE showed an inhibition of total and progressive motility on mouse sperm alongside a significant decrease of motility kinematic parameters and IVF rates. The results confirm the antifertility effect of this plant used in Mexican folk medicine. Further study on OBACE as a possible contraceptive treatment is warranted because of its activity and low in vivo toxicity. ABBREVIATIONS: ALH: lateral amplitude; AP: acid phosphatase; BCF: beat frequency; BSA: bovine serum albumine; CTC: chlortetracycline; FDA: fluorescein diacetate; Fura-2 AM: fura-2-acetoxymethyl ester; HIV: human immunodeficiency virus; IVF: in vitro fertilization; OBACE: aqueous crude extract of Echeveria gibbiflora; PI: propidum iodide; SN: supernatant; VAP: average path velocity; VCL: track speed; VSL: straight line velocity.

Bisphenol A accelerates capacitation-associated protein tyrosine phosphorylation of rat sperm by activating protein kinase A.

Bisphenol A (BPA) is a synthetic estrogen-mimic chemical. It has been shown to affect many reproductive endpoints. However, the effect of BPA on the mature sperm and the mechanism of its action are not clear yet. Here, our in vitro studies indicated that BPA could accelerate sperm capacitation-associated protein tyrosine phosphorylation in time- and dose-dependent manners. In vivo, the adult male rats exposed to a high dose of BPA could result in a significant increase in sperm activity. Further investigation demonstrated that BPA could accelerate capacitation-associated protein tyrosine phosphorylation even if sperm were incubated in medium devoid of BSA, HCO3 (-), and Ca(2+) However, this action of BPA stimulation could be blocked by H89, a highly selective blocker of protein kinase A (PKA), but not by KH7, a specific inhibitor of adenylyl cyclase. These data suggest that BPA may activate PKA to affect sperm functions and male fertility.

Two activators of in vitro fertilization in mice from licorice.

Systems for artificial insemination have been established in some animals. However, due to limited availability of sperm and oocytes, more effective treatment methodologies are required. Recently, it was demonstrated that the rate of in vitro fertilization (IVF) in mice was improved by adding a water extract of licorice (Glycyrrhiza uralensis), but not glycyrrhizic acid, to the artificial insemination culture medium. In this study, we examined licorice extract for active compounds using bioassay-guided separation. The results indicated that isoliquiritigenin and formononetin were the active molecules in licorice that contributed to the improved rate of IVF.

[Application of traditional Chinese medicine in IVF-ET].

The factors impacting the pregnancy rate and the live birth rate mainly include ovary function disorder and low endometrial receptivity, which can cause the difficulty in embryo implantation, early miscarriage and pregnancy failure. In recent years, researchers of the traditional Chinese medicine (TCM) have made active efforts in assisting IVF-ET, so as to achieve a great advance in improving the ovary reaction, treating the ovarian hyperstimulation syndrome (OHSS), improving the follicle, embryo quality and endometrial receptivity and protecting the fetus, which had been summarized in this article.

Effects of FGF10 on bovine oocyte meiosis progression, apoptosis, embryo development and relative abundance of developmentally important genes in vitro.

Fibroblast growth factor (FGF10) acts at the cumulus oocyte complex, increasing the expression of cumulus cell expansion-related genes and oocyte competency genes. We tested the hypothesis that addition of FGF10 to the maturation medium improves oocyte maturation, decreases the percentage of apoptotic oocytes and increases development to the blastocyst stage while increasing the relative abundance of developmentally important genes (COX2, CDX2 and PLAC8). In all experiments, oocytes were matured for 22 h in TCM-199 supplemented with 0, 2.5, 10 or 50 ng/ml FGF10. In Experiment 1, after maturation, oocytes were stained with Hoechst to evaluate meiosis progression (metaphase I, intermediary phases and extrusion of the first polar body) and submitted to the TUNEL assay to evaluate apoptosis. In Experiment 2, oocytes were fertilized and cultured to the blastocyst stage. Blastocysts were frozen for analysis of COX2, CDX2 and PLAC8 relative abundance. In Experiment 1, 2.5 ng/ml FGF10 increased (p < 0.05) the percentage of oocytes with extrusion of the first polar body (35%) compared to 0, 10 and 50 ng/ml FGF10 (21, 14 and 12%, respectively) and FGF10 decreased the percentage of oocytes that were TUNEL positive in all doses studied. In Experiment 2, there was no difference in the percentage of oocytes becoming blastocysts between treatments and control. Real-time RT-PCR showed a tendency of 50 ng/ml FGF10 to increase the relative abundance of COX2 and PLAC8 and of 10 ng/ml FGF10 to increase CDX2. In conclusion, the addition of FGF10 to the oocyte maturation medium improves oocyte maturation in vitro, decreases the percentage of apoptotic oocytes and tends to increase the relative abundance of developmentally important genes.

Nitric oxide synthase (NOS) inhibition during porcine in vitro maturation modifies oocyte protein S-nitrosylation and in vitro fertilization.

Nitric oxide (NO) is a molecule involved in many reproductive processes. Its importance during oocyte in vitro maturation (IVM) has been demonstrated in various species although sometimes with contradictory results. The objective of this study was to determine the effect of NO during IVM of cumulus oocyte complexes and its subsequent impact on gamete interaction in porcine species. For this purpose, IVM media were supplemented with three NOS inhibitors: NG-nitro-L-arginine methyl ester (L-NAME), NG-monomethyl-L-arginine (L-NMMA) and aminoguanidine (AG). A NO donor, S-nitrosoglutathione (GSNO), was also used. The effects on the cumulus cell expansion, meiotic resumption, zona pellucida digestion time (ZPdt) and, finally, on in vitro fertilization (IVF) parameters were evaluated. The oocyte S-nitrosoproteins were also studied by in situ nitrosylation. The results showed that after 42 h of IVM, AG, L-NAME and L-NMMA had an inhibitory effect on cumulus cell expansion. Meiotic resumption was suppressed only when AG was added, with 78.7% of the oocytes arrested at the germinal vesicle state (P<0.05). Supplementation of the IVM medium with NOS inhibitors or NO donor did not enhance the efficiency of IVF, but revealed the importance of NO in maturation and subsequent fertilization. Furthermore, protein S-nitrosylation is reported for the first time as a pathway through which NO exerts its effect on porcine IVM; therefore, it would be important to determine which proteins are nitrosylated in the oocyte and their functions, in order to throw light on the mechanism of action of NO in oocyte maturation and subsequent fertilization.

Tanshinone IIA exhibits anticonvulsant activity in zebrafish and mouse seizure models.

Danshen or Chinese red sage (Salvia miltiorrhiza, Bunge) is used by traditional Chinese medicine (TCM) practitioners to treat neurological, cardiovascular, and cerebrovascular disorders and is included in some TCM formulations to control epileptic seizures. In this study, acetonic crude extracts of danshen inhibited pentylenetetrazol (PTZ)-induced seizure activity in zebrafish larvae. Subsequent zebrafish bioassay-guided fractionation of the extract resulted in the isolation of four major tanshinones, which suppressed PTZ-induced activity to varying degrees. One of the active tanshinones, tanshinone IIA, also reduced c-fos expression in the brains of PTZ-exposed zebrafish larvae. In rodent seizure models, tanshinone IIA showed anticonvulsive activity in the mouse 6-Hz psychomotor seizure test in a biphasic manner and modified seizure thresholds in a complex manner for the mouse i.v. PTZ seizure assay. Interestingly, tanshinone IIA is used as a prescription drug in China to address cerebral ischemia in patients. Here, we provide the first in vivo evidence demonstrating that tanshinone IIA has anticonvulsant properties as well.

Endocrine effects of hCG supplementation to recombinant FSH throughout controlled ovarian stimulation for IVF: a dose-response study.

OBJECTIVE: To analyse the endocrine response in relation to the Δ-4 and Δ-5 pathways of ovarian steroidogenesis after different doses of human chorionic gonadotrophin (hCG) supplementation to recombinant FSH from Day 1 of controlled ovarian stimulation for IVF. DESIGN: A randomized dose-response pilot study. PATIENTS: A total of 62 IVF patients aged 25-37 years with regular cycles and FSH <12 IU/l were treated with a fixed dose of rFSH 150 IU/day and randomized to four hCG dose groups: Dose 0: 0 IU/day, Dose 50: 50 IU/day, Dose 100: 100 IU/day and Dose 150: 150 IU/day. RESULTS: A significant hCG dose-dependent incremental increase was found for progesterone (49-160%), 17-OH-progesterone (223-614%), androstenedione (91-340%) and testosterone (95-338%) from Dose 0 to Dose 150, respectively. Dehydroepiandrosterone (DHEA) showed minor changes during stimulation and no differences between the groups. The highest oestradiol concentrations were observed in Dose 100 and Dose 150. Sex hormone-binding globulin (SHBG) increased similarly in all groups at the end of stimulation. No difference was observed for anti-müllerian hormone (AMH) concentration between the groups, but a 50% decline from the start to the end of the stimulation was found. CONCLUSION: Supplementation with hCG resulted in a clear dose-related response for androgens, progesterone and 17-OH-progesterone. Oestradiol concentration reached maximum levels with an hCG dose of 100 IU/day, suggesting saturation of aromatase function. No difference between the groups was observed for DHEA, supporting that the stimulatory effects of hCG doses on androgens and oestrogen production were mainly induced via the Δ-5 pathway. SHBG, being a biomarker of oestrogen/androgen balance, was not changed by increasing hCG.

The effects of N-acetyl-L-cysteine supplementation on in vitro porcine oocyte maturation and subsequent fertilisation and embryonic development.

The effects of supplementation with 1.5 mM n-acetyl-l-cysteine (NAC) during in vitro oocyte maturation were studied. Oocytes were supplemented with 1.5 mM NAC during maturation for 0 to 24 h, 24 to 48 h, or 0 to 48 h then subjected to IVF and embryo development. Oocytes were evaluated after maturation for intracellular glutathione concentration, superoxide dismutase and glutathione peroxidase activities and DNA fragmentation. Fertilisation and embryonic development success were also evaluated. There was no effect of treatment on intracellular glutathione concentrations, enzyme activities or fertilisation success rates. Supplementing NAC during maturation significantly decreased (P < 0.05) the percentage of oocytes with fragmented DNA compared with no NAC supplementation. Supplementing NAC from 24 to 48 h or 0 to 48 h resulted in a significantly higher (P < 0.05) percentage of oocytes with male pronuclei than for oocytes from the other treatment groups. There was no difference in the percentage of embryos cleaved by 48 h after IVF between treatment groups. Supplementing NAC from 24 to 48 h or 0 to 48 h resulted in a significantly higher (P < 0.05) percentage of embryos reaching the blastocyst stage by 144 h after IVF compared with the other treatment groups. These results indicate that supplementation of the oocyte maturation medium with 1.5 mM NAC, specifically during the last 24 h, improves male pronucleus formation and blastocyst development in pigs.

Endoplasmic reticulum (ER) stress in cumulus-oocyte complexes impairs pentraxin-3 secretion, mitochondrial membrane potential (DeltaPsi m), and embryo development.

Fatty acids such as palmitic acid at high levels are known to induce endoplasmic reticulum (ER) stress and lipotoxicity in numerous cell types and thereby contribute to cellular dysfunctions in obesity. To understand the impact of high fatty acids on oocytes, ER stress and lipotoxicity were induced in mouse cumulus-oocyte complexes during in vitro maturation using the ER Ca(2+) channel blocker thapsigargin or high physiological levels of palmitic acid; both of which significantly induced ER stress marker genes (Atf4, Atf6, Xbp1s, and Hspa5) and inositol-requiring protein-1α phosphorylation, demonstrating an ER stress response that was reversible with the ER stress inhibitor salubrinal. Assessment of pentraxin-3, an extracellular matrix protein essential for fertilization, by immunocytochemistry and Western blotting showed dramatically impaired secretion concurrent with ER stress. Mitochondrial activity in oocytes was assessed by 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide staining of inner mitochondrial membrane potential, and oocytes matured in thapsigargin or high-dose palmitic acid had significantly reduced mitochondrial activity, reduced in vitro fertilization rates, and were slower to develop to blastocysts. The deficiencies in protein secretion, mitochondrial activity, and oocyte developmental competence were each normalized by salubrinal, demonstrating that ER stress is a key mechanism mediating fatty acid-induced defects in oocyte developmental potential.

[Effects of inositol on oocyte quality in patients affected with polycystic ovary syndrome]. / Effetti dell'inositolo sulla qualità ovocitaria in pazienti affette da sindrome dell'ovaio policistico.

AIM: Polycystic ovary syndrome is the most common cause of chronic anovulation infertility in women in fertile period. The supplementation of inositol, due to its ability to increase insulin sensitivity, improves the oocytes' quality and increase the number of oocytes collected after ovarian stimulation in patients undergoing IVF (In Vitro Fertilization). The aim of our study is to determine the effects of myo-inositol on oocyte's quality on a sample of women with polycystic ovary syndrome. METHODS: The patients were divided into two groups: patients of Group A intook 2 g of myo-inositol + 400 µg of folic acid 2 times a day, continuously for 3 months, while Group B only 400 µg of folic acid. RESULTS AND CONCLUSION: At the end of treatment, the number of follicles of diameter >15 mm, visible at ultrasound during stimulation, and the number of oocytes recovered at the time of pick-ups were found to be significantly greater in the group treated with myo-inositol, so as the average number of embryos transferred and embryo Grade G1. Significantly reduced was the average number of immature oocytes (vesicles germ and degenerated oocytes) too.