Exploring the Role of Hyaluronic Acid in Reproductive Biology and Beyond: Applications in Assisted Reproduction and Tissue Engineering.

Hyaluronic acid (HA) plays a prominent role in various aspects of reproductive biology and assisted reproductive technologies (ART). This review describes the multifaceted influence of HA, ranging from primordial germ cell migration, ovarian follicle development, and ovulation in females to sperm structure, physiology, motility, and capacitation in males. In addition, HA also plays an important role in fertilization and promotes embryo implantation by mediating cellular adhesion and communication within the uterus. Against this physiological background, the review examines the current applications of HA in the context of ART. In addition, the article addresses the emerging field of reproductive tissue engineering, where HA-based hydrogels offer promising perspectives as they can support the development of mature oocytes and spermatogenesis in vitro. Overall, this review highlights the integral role of HA in the intricate mechanisms of reproductive biology and its growing importance for improving ART outcomes and the field of tissue engineering of the reproductive system.

Seminal Oxidative Stress and Sperm DNA Fragmentation in Men from Couples with Infertility or Unexplained Recurrent Pregnancy Loss.

(1) Background: This case-control study examined whether men from couples with unexplained recurrent pregnancy loss (RPL) or infertility exhibited higher seminal oxidative stress (OS) and sperm DNA fragmentation (SDF) compared to fertile controls. (2) Methods: The study included 30 participants from each group: unexplained RPL, unexplained infertility, and proven fertility. Data were collected at Aalborg University Hospital tertiary RPL and fertility treatment clinics (Aalborg, Denmark), excluding couples with mixed conditions for homogeneity. Semen samples were analyzed using computer-aided sperm analysis (CASA) for concentration, motility, and morphology. SDF was assessed via a CASA-based sperm chromatin dispersion test. OS was measured as static oxidation-reduction potential (sORP). (3) Results: The results showed no significant OS differences between groups. The RPL group had significantly lower SDF levels than the control group. A significant positive correlation between SDF and OS was observed in the infertility group. Overall, this study did not find significant differences in OS levels between men from couples with unexplained RPL or infertility and fertile controls, while SDF levels were lower in the RPL group compared to controls. (4) Conclusion: In conclusion, despite the existing literature suggesting that OS and SDF are negative prognostic factors, our findings suggest they may not be reliable diagnostic markers for RPL and infertility.

Modulatory Effect of Probiotic Lactobacillus rhamnosus PB01 on Mechanical Sensitivity in a Female Diet-Induced Obesity Model.

Obese animals and humans demonstrate higher sensitivity to pain stimuli. Among the endogenous factors prompting obesity, the intestinal microbiota has been proposed to influence responsiveness to pain. The beneficial effects of probiotics on obesity are well documented, whereas data on their analgesic efficacy is minimal. The protective effect of probiotics on nociception in diet-induced obese male mice has been previously demonstrated, but the sex differences in pain sensitivity and analgesic response do not allow for the generalization of these findings to the female gender. Hence, this study aimed at investigating the potential effects of oral probiotic supplementation on mechanical pain thresholds in female diet-induced obese mice compared with controls. Thirty-two adult female mice (N=32) were randomly divided into two groups receiving standard (normal-weight group; NW) or high-fat diet (diet-induced obesity; DIO). All rats received a single daily dose (1 × 109 CFU) of probiotics (Lactobacillus rhamnosus PB01, DSM14870) for four weeks by gavage. Mechanical pain thresholds were recorded by an electronic von Frey device at baseline, at the end of weeks 2, 4, 6, and 8 in both DIO and NW groups with and without consumption of probiotics. Blood samples were obtained for the measurement of lipid profile and reproductive hormone levels. Bodyweight was considerably lower (P < 0.001) in groups supplied with probiotics than groups without probiotics. Pressure pain threshold values showed a significant (P < 0.001) increase (reduced pain sensitivity) following probiotic supplementation, proposing a modulatory effect of probiotics on mechanical sensory circuits and mechanical sensitivity, which might be a direct consequence of weight loss or an indirect result of the probiotics' anti-inflammatory properties. Understanding the precise underlying mechanism for the effect of probiotics on weight loss and mechanical pain sensitivity seen in this study warrants further investigation.

Comparison of commercially available chamber slides for computer-aided analysis of human sperm.

Despite the increasing use of computer-aided sperm analysis (CASA) in clinical practice, there is still no golden standard for the type of slide to be used with these systems. Counting chamber depth and loading method, can profoundly influence motility and concentration estimates, thereby undermining the validity and accuracy of CASA. To contribute toward standardized sperm analysis, this study compared different commercially available capillary-filled slides including 10 and 20 µm deep Leja slides (Leja10 and Leja20); 10, 16 and 20 µm deep CellVision slides (CV10, CV16 and CV20); and drop-loaded slides including slide and coverslip (SCS) with a depth of 20.1 µm and the Makler chamber with a depth of 10 µm for sperm analysis when using CASA. The Sperm Class Analyzer (SCA) CASA system was used to assess concentration, motility, and detailed kinematic parameters of 20 normozoospermic human samples using the different chamber slides. Results were evaluated by the repeated measures ANOVA and Intraclass correlation coefficients. The Makler chamber showed significantly (P < 0.05) higher concentrations than other slides. However, there was no significant difference in the percentage of sperm in different motility groups among the slides. CV10, Leja10 and Makler showed significantly higher curvilinear-, average path- and straight-line velocity (VCL, VAP, VSL) values than other slides. In conclusion, despite the objectiveness of the assessments by CASA systems, there are still some discrepancies in the results of sperm concentration, motility and other kinematic parameters when using different commercially available slides. The possible negative influence of the sperm quality misdiagnosis on the selection of treatment strategy in a clinical setting, emphasizes the need for further standardization and quality control of the commercially available chamber slides for use with CASA. Furthermore, this study found more consistent results for capillary-filled chambers compared to drop-loaded slides, suggesting a superior method when using CASA.Abbreviations: DNA: Deoxyribonucleic acid; CASA: Computer-aided semen analysis; SCA: Sperm Class Analyzer; WHO: World Health Organization; CV: CellVision; SCS: Slide and Coverslip; SD: Standard deviation; ANOVA: Analysis of Variance; PR: Progressive; NP: Non-progressive; IMT: Immotile; VCL: Curvilinear velocity; VAP: Average path velocity; VSL: Straight-line velocity; Lin: Linearity; STR: Straightness; WOB: Wobble; ALH: Amplitude of lateral head displacement; BCF: Beat cross frequency.

Effect of Maternal Age on Hippo Pathway Related Gene Expressions and Protein Localization Pattern in Human Embryos.

OBJECTIVE: The Hippo pathway plays an important role in embryo development, and separation of trophectoderm (TE) and inner cell mass (ICM) cell lines. Therefore, this study investigated effect of maternal age on activity of Hippo pathway in human embryos. MATERIALS AND METHODS: In this experimental study, the developed up embryos to the blastocyst stage and the embryos whose growth stopped at the morula stage were collected from women aged 20-30 years old (young group, 94 embryos) and >37 years (old group, 89 embryos). Expression of OCT4, SOX2, CDX2, GATA3, YAP genes and the relevant proteins, in the both groups were evaluated using respectively quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and immunofluorescence methods. RESULTS: There was no significant difference in the expression level of OCT4, SOX2, CDX2, GATA3 and YAP genes in blastocyst and morula stages, between the two groups. However, SOX2 and CDX2 gene expressions in morula stage embryos of the old group was statistically lower than that of the young group (P=0.007 and P=0.008, respectively). Additionally, in the embryos collected from women with >37 years of age, at the blastocyst stage, phospho-YAP (p-YAP) protein was found to be accumulated in the TE, but it was almost disappeared from the ICM. Additionally, in the old group, contrary to the expectation, YAP protein was expressed in the ICM, rather than TE. CONCLUSION: The results of this study showed that YAP and P-YAP among the Hippo signalling pathway may be altered by increasing age.

Evolution of ASC Immunophenotypical Subsets During Expansion In Vitro.

Adipose-derived stromal/stem cells (ASCs) are currently being considered for clinical use for a number of indications. In order to develop standardized clinical protocols, it is paramount to have a full characterization of the stem cell preparations. The surface marker expression of ASCs has previously been characterized in multiple studies. However, most of these studies have provided a cross-sectional description of ASCs in either earlier or later passages. In this study, we evaluate the dynamic changes of 15 different surface molecules during culture. Using multichromatic flow cytometry, ASCs from three different donors each in passages 1, 2, 4, 6, and 8 were analyzed for their co-expression of markers associated with mesenchymal stem cells, wound healing, immune regulation, ASC markers, and differentiation capacity, respectively. We confirmed that at an early stage, ASC displayed a high heterogeneity with a plethora of subpopulations, which by culturing became more homogeneous. After a few passages, virtually all ASCs expressed CD29, CD166 and CD201, in addition to canonical markers CD73, CD90, and CD105. However, even at passage 8, there were several predominant lineages that differed with respect to the expression of CD34, CD200 and CD271. Although the significance of remaining subpopulations still needs to be elucidated, our results underscore the necessity to fully characterize ASCs prior to clinical use.

Total Antioxidant Capacity; A Potential Biomarker for Non-Invasive Sex Prediction in Culture Medium of Preimplantation Human Embryos.

OBJECTIVE: The presence of a sex related metabolic difference in glucose utilization and, on the other hand, different developmental kinetic rates in human preimplantation embryos, has been previously observed, however, the correlation between these two events is unknown. Oxidative stress (OS) induced by higher glucose consumption appears to be a possible cause for the delayed development rate in female embryos. We examined the correlation between glucose consumption and total antioxidant capacity (TAC) concentration in individual embryo culture media for both male and female embryos. MATERIALS AND METHODS: In this cross-sectional study, we evaluated high quality embryos from 51 patients that underwent intracytoplasmic sperm injection (ICSI) and preimplantation genetic diagnosis (PGD) at the Royan Institute between December 2014 and September 2017. The embryos were individually cultured in G-2TM medium droplets at days 3-5 or 48 hours post PGD. We analysed the spent culture media following embryo transfer for total antioxidant capacity (TAC) and any remaining glucose concentrations through fluorometric measurement by chemiluminecence system which indirectly was used for measurement of glucose consumed by embryos. RESULTS: The results showed that female embryos consumed more glucose which was associated with decreased TAC concentration in their culture medium compared to male embryos. The mean of glucose concentration consumed by the female embryos (30.7 ± 4.7 pmol/embryo/hour) was significantly higher than that of the male embryos (25.3 ± 3.3 pmol/embryo/hour) (P<0.001). There were significantly lower levels of TAC in the surrounding culture medium of female embryos (22.60 ± 0.19 nmol/µl) compared with male embryos (24.74 ± 0.27 nmol/µl, P<0.01). CONCLUSION: This finding highlighted the utilization of sex dependent metabolic diversity between preimplantation embryos for non-invasive sex diagnosis and suggests the TAC concentration as a potential noninvasive biomarker for prediction of sex.

Lactobacillus rhamnosus PB01 (DSM 14870) supplementation affects markers of sperm kinematic parameters in a diet-induced obesity mice model.

Probiotics have been proposed as alternatives to pharmacological products in several medical conditions including the modulation of obesity, which is frequently associated with poor semen quality. However, effects of probiotics on male fertility have been less investigated. This study assessed the effect of Lactobacillus rhamnosus PB01 (DSM-14870) on sperm kinematic parameters in Normal-weight (NW) and diet-induced obese (DIO) models. NW and DIO C57BL/6NTac mice were divided into two subgroups with or without a single daily dose (1x109CFU) of L. rhamnosus for four weeks. Sperm motility and kinematics together with blood lipid profiles and reproductive hormone levels were assessed using the sperm class analyzer system. Probiotic supplementation increased serum testosterone, LH and FSH levels in both NW and DIO groups resulting in significantly (P<0.05) higher velocity (VSL, VCL and VAP) and percentages of progressively motile sperm and significantly lower percentages of immotile sperm. Other kinematic parameters (Lin, STR, ALH and BCF) were also increased in both probiotic supplemented DIO and NW groups at the 10% level of significance. Probiotic supplemented DIO mice demonstrated significantly higher percentages of progressively motile sperm versus DIO controls. This study demonstrated the potential of L. rhamnosus PB01 as a regulatory agent with positive effects on weight loss and reproductive-hormones, significantly improving sperm motility and kinematic parameters in male DIO models.

Effects of Chronic Musculoskeletal Pain on Fertility Potential in Lean and Overweight Male Patients.

Both chronic pain and obesity are known to affect reproductive hormone profiles in male patients. However, the effect of these conditions, alone or in combination, on male fertility potential has received less attention. 20 chronic musculoskeletal pain patients and 20 healthy controls were divided into lean and overweight subgroups according to their BMI. Current level of chronic pain (visual analogue scale) and pressure pain thresholds (PPTs) in 16 predefined sites, classically described and tested as painful points on the lower body, were measured. Levels of reproductive hormone and lipid profiles were assessed by ELISA. Sperm concentration and motility parameters were analyzed using a computer-aided sperm analysis system. Sperm concentration, progressive motility, and percentage of hyperactivated sperm were generally lower in the chronic pain patients in both lean and overweight groups. The overweight control and the lean chronic pain groups demonstrated a significantly lower percentage of progressively motile sperm compared with the lean control group, suggesting that musculoskeletal chronic pain may have a negative influence on sperm quality in lean patients. However, due to the potential great negative influence of obesity on the sperm parameters, it is difficult to propose if musculoskeletal chronic pain also influenced sperm quality in overweight patients. Further research in chronic pain patients is required to test this hypothesis.

Potential Nociceptive Regulatory Effect of Probiotic Lactobacillus rhamnosus PB01 (DSM 14870) on Mechanical Sensitivity in Diet-Induced Obesity Model.

Treatments for obesity have been shown to reduce pain secondary to weight loss. Intestinal microbiota, as an endogenous factor, influences obesity and pain sensitivity but the effect of oral probiotic supplementation on musculoskeletal pain perception has not been studied systematically. The present study examined the effect of a single daily oral dose (1 × 10(9) CFU) of probiotics (Lactobacillus rhamnosus PB01, DSM14870) supplement on mechanical pain thresholds in behaving diet-induced obese (DIO) mice and their normal weight (NW) controls. The mice (N = 24, 6-week-old male) were randomly divided into four groups on either standard or high fat diet with and without probiotic supplementation. Both DIO and NW groups with probiotic supplementation maintained an insignificant weight gain while the control groups gained significant weight (P < 0.05). Similarly, both DIO and NW probiotics supplemented groups demonstrated a significantly (P < 0.05) lower sensitivity to mechanical stimulation compared to their corresponding control. The results of this study suggest a protective effect of probiotics on nociception circuits, which propose a direct result of the weight reduction or an indirect result of anti-inflammatory properties of the probiotics. Deciphering the exact underlying mechanism of the weight loss and lowering nociception effect of the probiotic applied in this study require further investigation.

Quantitative expression of developmental genes, Pou5f1 (Oct4) and Mest (Peg1), in vitrified mouse embryos.

BACKGROUND: Embryo cryopreservation is the process that water is removed from the cell by cryoprotectant materials, and embryos are stored at temperature below zero. This process may affect the viability and developmental potential of embryos. OBJECTIVE: In this study, the effect of the vitrification cryotop method on the expression level of Oct4 and Mest developmental genes in mouse blastocysts was examined. MATERIALS AND METHODS: The collected 2-cell embryos of superovulated mouse by oviduct flushing were divided into non-vitrified and vitrified groups. These embryos were cultured to the blastocyst stage directly in the non-vitrified group and in the vitrified group, these embryos were cultured to 4-8 cell embryos, vitrified with cryotop in these stages and after 2-6 months, warmed and cultured to blastocyst embryos. Quantitative expression of two developmental genes, namely Oct4 and Mest, were performed in these groups, using RNA purification and Real-time RT-PCR. RESULTS: Quantitative PCR analysis showed that the expression level of both genes, Oct4 and Mest, was reduced significantly in the vitrified-warmed group relative to the control group (p=0.046 and p=0.001). CONCLUSION: This study revealed that morphologically normal embryos show a reduced amount of Oct4 and Mest transcripts which indicate that the vitrification method negatively effects the expression level of these two developmental genes.

Development of 4-cell mouse embryos after re-vitrification.

This paper reports studies on the effects of re-vitrification by the CPS (Closed Pulled Straw) method on the development of 4-cell stage mouse embryos. The procedure involved culturing 2-cell mouse embryos in G-1 medium until the 4-cell stage followed by the division of the normal 4-cell stage embryos into a control group (non-vitrified) and two experimental subgroups (vitrified and re-vitrified). Embryos in the vitrified subgroup were cryopreserved by the CPS vitrification method. In the second experimental subgroup (re-vitrified), embryos that were already vitrified were warmed and cryopreserved again by the same method. There was no significant reduction in the rate of blastocyst formation after vitrification and re-vitrification. However, re-vitrification reduced the total cell number, ICM (inner cell mass) percent and blastocyst diameter (P<0.05). These results showed that vitrification and re-vitrification by the CPS method did not negatively affect the development of vitrified-warmed 4-cell mouse embryos, whereas re-vitrification significantly reduced both the cell number and diameter of blastocysts.

Fatty Acid Prof iles of Ram's Sperm after Removing Some Fatty Acid Sources from the Diets and Persistency of Fatty Acids in Sperm.

BACKGROUND: Mammalian spermatozoa are characterized by a high proportion of polyunsaturated fatty acids (PUFAs), but reliable data concerning dietary effects on fatty acid (FA) profile in ram's sperm and the persistency of FA in the ration to the FA in sperm has not been reported. Therefore, the aim of this study was to determine the stability of saturated and unsaturated FAs in ram's sperm despite removing FA sources from their diet. MATERIALS AND METHODS: Nine Kalkoohi rams were used in a completely randomized design and they were assigned to 3 groups. The treatments were diet supplemented (35 g/d/ram) by C16:0 (RP-10®), C18: 2 (Sunflower oil; SO) and n-3 (Fish oil; FO) with Vitamin E. Fifteen weeks after the start of the supplemented diet, rams were offered a basal diet without any supplementary FA source for 35 days when the sperm's FA ratio was determined. The data were analyzed by ANOVA (Analysis of variance) using the General Linear Model (GLM) procedure of SAS Institute. RESULTS: THIRTY FIVE DAYS AFTER REMOVING THE FAT SUPPLEMENT FROM THE DIET, MAJOR FA IN SPERM CONSISTED OF: C14:0, C16:0, C18:0, C18:1 cis, C18:2 cis and C22:6 n-3 docosahexaenoic acid (DHA). The percentage of C14:0 (p=0.8) and C18:1 cis (P =0.4) were similar among all the treatments. Interestingly, 35 days after the removal of fatty acid source, the percentage of C22:6 was highest in the FO treated group. CONCLUSION: The different sperm FA profile among various groups suggests that dietary FA had significant direct or indirect impacts on sperm FA profile after 35 days which might lead to physical and chemical changes in sperm characteristics.

Achieving high survival rate following cryopreservation after isolation of prepubertal mouse spermatogonial cells.

PURPOSE: Isolating spermatogonia cells with high purity and viability and achieving better survival rate following cryopreservation METHODS: Isolating the cells by Magnetic Activating Cell Sorting (MACS) method using anti CD49f (alpha6 integrin) antibody and Dynabeads and freezing in DMSO-based freezing mediums containing three different FBS concentrations of 50%, 60% and 70%. RESULTS: The mean (+/-SD) purity of the isolated cells was 92.52+/-3.57 (range 92.43-98.25). The cells frozen in group I, II and III had mean 39.60+/-1.48 (range 37.98-41.62), 89.05+/-3.83 (range 80.83-90.33) and 90.52+/-1.71 (range 89.07-92.52) viability, respectively. CONCLUSION: Higher viable cell counts and purity can be attained by the use of alpha6 integrin and magnetic beads. After the thawing of spermatogonial cells, optimum viability was achieved in freezing media containing 60% FBS.