Survival and hormone production of isolated mouse follicles in three-dimensional artificial scaffolds after stimulation with bpV(HOpic).

PURPOSE: To preserve fertility before gonadotoxic therapy, ovarian tissue can be removed, cryopreserved, and transplanted back again after treatment. An alternative is the artificial ovary, in which the ovarian follicles are extracted from the tissue, which reduces the risk of reimplantation of potentially remaining malignant cells. The PTEN inhibitor bpV(HOpic) has been shown to activate human, bovine and alpacas ovarian follicles, and it is therefore considered a promising substance for developing the artificial ovary. The purpose of this study was to examine the impact of different scaffolds and the vanadate derivative bpV(HOpic) on mice follicle survival and hormone secretion over 10 days. METHODS: A comparative analysis was performed, studying the survival rates (SR) of isolated mice follicle in four different groups that differed either in the scaffold (polycaprolactone scaffold versus polyethylene terephthalate membrane) or in the medium-bpV(HOpic) versus control medium. The observation period of the follicles was 10 days. On days 2, 6, and 10, the viability and morphology of the follicles were checked using fluorescence or confocal microscopy. Furthermore, hormone levels of estrogen (pmol/L) and progesterone (nmol/L) were determined. RESULTS: When comparing the SR of follicles among the four groups, it was observed that on day 6, the study groups utilizing the polycaprolactone scaffold with bpV(HOpic) in the medium (SR: 0.48 ± 0.18; p = 0.004) or functionalized in the scaffold (SR: 0.50 ± 0.20; p = 0.003) exhibited significantly higher survival rates compared to the group using only the polyethylene terephthalate membrane (SR: 0). On day 10, a significantly higher survival rate was only noted when comparing the polycaprolactone scaffold with bpV(HOpic) in the medium to the polyethylene terephthalate membrane group (SR: 0.38 ± 0.20 versus 0; p = 0.007). Higher levels of progesterone were only significantly associated with better survival rates in the group with the polycaprolactone scaffold functionalized with bpV(HOpic) (p = 0.017). CONCLUSION: This study demonstrates that three-dimensional polycaprolactone scaffolds improve the survival rates of isolated mice follicles in comparison with a conventional polyethylene terephthalate membrane. The survival rates slightly improve with added bpV(HOpic). Furthermore, higher rates of progesterone were also partly associated with improved survival.

Individual dynamics of uterine natural killer cells in natural and stimulated cycles monitored using a new endometrial dating method.

PROBLEM: It is important to evaluate the dynamics of uterine natural killer (uNK) cells in hormone replacement therapy (HRT) cycles, given their potential role in implantation and the common usage of HRT cycles with in vitro fertilization (IVF). METHOD OF STUDY: A total of 132 subfertile patients were evaluated during the secretory phase of either natural ovulation (OV) or HRT cycles, with two biopsies taken on approximately days 5 and 10 after ovulation/progesterone administration in a single menstrual cycle. Immunohistochemical Personal Endometrial Maturation Analysis (PEMA) was used to better quantify secretory-phase endometrial development, in combination with subsequent evaluation of uNK cell density. RESULTS: uNK cell density increased rapidly from the early to mid-secretory phase, with mean uNK densities of 113 and 117 per mm2 in first biopsies and 315 and 387 per mm2 in second biopsies for OV and HRT cycles, respectively. After reassessment of endometrial development with PEMA, the first and second biopsies in HRT and OV cycles were histologically dated to developmental ranges between days 15-20 (first biopsy) and days 19-25 (second biopsy). CONCLUSION: Subfertile women showed variable endometrial development in PEMA assessment, with uNK cell density correlating with the dating results. Overall, comparable levels of uNK cell density were observed in OV and HRT cycles. Importantly, uNK cell density depends on the histological maturation stage, with similar low coefficients of determination. This observation suggests that aberrant uNK cell results more likely reflect displaced endometrial maturation, rather than an intrinsic anomaly in uNK cell trafficking.

Endometrial delay is found to be part of a normal individual dynamic transformation process.

PURPOSE: Limited information is clinically available concerning endometrial receptivity; assessing endometrial transformation status is therefore an urgent topic in assisted reproductive technology. This study aimed to investigate individual endometrial transformation rates during the secretory phase in subfertile patients using personal endometrial transformation analysis. METHODS: Monitoring was carried out during the secretory phase to obtain endometrial receptivity profiles. For the investigation, two endometrial biopsies were taken within one menstrual cycle. The extended endometrial dating was based on the Noyes criteria, combined with immunohistochemical analyses of hormone receptors and proliferation marker Ki-67. Biopsies were taken mainly at days ovulation (OV, n = 76)/hormone replacement therapy (HRT, n = 58) + 5 and + 10. RESULTS: The results of the two biopsies were correlated with the clinically expected day of the cycle and showed temporal delays or hypercompensations, diverging from the expected cycle days by 0.5-5 days. In comparison with the first biopsies, the transformation rate in the second biopsies showed compensation, augmented delay, or constant transformation in 48.69, 22.37, and 28.94% of cases for ovulation in natural cycles and 56.89, 25.85, and 17.26% for HRT cycles, respectively. CONCLUSION: The study revealed an individually dynamic transformation process of the endometrium, with the ability to compensate or enlarge an initial "delay", which is now identified as a normal individual transformation process during the secretory phase. This information is of great importance for the scientific investigation of dynamic changes in endometrial tissue, as well as for the timing of embryo transfers.

Cell-based endometrial regeneration: current status and future perspectives.

Endometrial-related disorders including Asherman's syndrome, thin endometrium, pelvic organ prolapse, and cesarean scar pregnancies can be accompanied by different symptoms such as amenorrhea, infertility, abnormal placental implantation and recurrent miscarriage. Different methods have been introduced to overcome these problems such as surgery and hormonal therapy but none of them has shown promising outcomes. On the other hand, the development of novel regenerative therapeutic strategies has opened new avenues for the treatment of endometrial-related deficiencies. In this regard, different types of scaffolds, acellular matrices and also cell therapy with adult or stem cells have been investigated for the treatment of endometrial-related deficiencies. In this paper, we review the current status of cell-based endometrium regeneration using scaffold dependent and scaffold-free methods and future perspectives in this field. Moreover, we discuss the endometrial diseases that can be candidates for cell-based treatments. Also, the cells with the potential for endometrial regeneration are explained.

Role of adipokines in the ovarian function: Oogenesis and steroidogenesis.

Adipokines are mainly produced by adipose tissue; however, their expression has been reported in other organs including female reproductive tissues. Therefore, adipokines have opened new avenues of research in female fertility. In this regard, studies reported different roles for certain adipokines in ovarian function, although the role of other recently identified adipokines is still controversial. It seems that adipokines are essential for normal ovarian function and their abnormal levels could be associated with ovarian-related disorders. The objective of this study is to review the available information regarding the role of adipokines in ovarian functions including follicular development, oogenesis and steroidogenesis and also their involvement in ovary-related disorders.

Me2SO perfusion time for whole-organ cryopreservation can be shortened: Results of micro-computed tomography monitoring during Me2SO perfusion of rat hearts.

Cryopreservation of whole organs and specific tissues is an important and continually expanding field of medicine. The protocols currently used for organ preservation do not ensure survivability and functionality; the protocols for ovarian tissue lead to acceptable outcomes, but these are still capable of further improvement. In general, cryopreservation protocols need to be optimized. One important approach to improving cryopreservation protocols in general involves reducing exposure to cytotoxic cryoprotective agents prior to freezing. This study, therefore, evaluated the real-time tissue penetration of dimethyl sulfoxide, a cryoprotective agent that is widely used in cryopreservation. Dimethyl sulfoxide penetration in rat hearts perfused with a 15% (v/v) dimethyl sulfoxide solution was examined in real-time using dynamic contrast-enhanced micro-computed tomography imaging. Viability of cardiomyocytes was not significantly affected by the dimethyl sulfoxide perfusion procedure. Two different perfusion rates were evaluated and compared with perfusion using a common iodine-based contrast agent (iomeprol). The dynamic contrast-enhanced micro-computed tomography imaging data showed that dimethyl sulfoxide flushes both the extracellular and intracellular spaces in rat heart tissue to 95% equilibration after ≈ 35 s via perfusion. Subsequent wash-out via perfusion is completed to 95% within ≈ 49 s. The equilibration duration routinely used in dimethyl sulfoxide-based protocols for cryopreservation should therefore be questioned. Shorter incubation duration would perhaps be sufficient, as well as being beneficial in relation to cell survivability. It would be helpful to have techniques for non-invasive real-time monitoring of the penetration of cryoprotective agents and such techniques should be used to revise cryopreservation protocols. Switching to perfusion-based equilibration procedures might be beneficial, if feasible.

Endometrial Dating Method Detects Individual Maturation Sequences During the Secretory Phase.

BACKGROUND/AIM: This study assessed whether a new immunohistochemical dating method allows precise endometrial dating allowing optimal timing for embryo transfer. PATIENTS AND METHODS: A novel method was used for endometrial dating, with parameters including menstrual cycle days, Noyes histological criteria, along with immunohistochemical expression pattern of estrogen and progesterone receptors and proliferation marker Ki-67. Endometrial maturation was analyzed on days +5 to +10 after ovulation or progesterone administration in 217 biopsies from 151 subfertile patients during the secretory phase. RESULTS: Endometrial maturation varied individually, occurring 1.68±1.67 days late. Comparison of histological maturation with clinical days after ovulation showed a delay of about 2 days. CONCLUSION: Endometrial maturation requires 8 days, rather than the expected 6 days, to reach the histological mid-secretory phase. This is not a delay and is also seen in fertile patients. The new analysis method used is superior to that using Noyes criteria alone and provides a better basis for determining conditions for optimal timing of embryo transfers.

Effects of bacteria on male fertility: Spermatogenesis and sperm function.

Bacterial infection can negatively affect different parts of the male genital tract and subsequently cause impaired spermatogenesis and male fertility. However, most of the previous studies have focused on the infected organs of the male genital tract and there are not many studies that investigated the direct effect of bacteria on sperm and their mechanism of action. Interestingly, bacteria can induce different damages on sperm cells such as DNA fragmentation, cell membrane peroxidation, and acrosome impairment. Such negative effects can be mediated by bacteria-secreted toxins and metabolites or by direct attachment of bacteria on the sperm cells and subsequent activation of signaling pathways related to oxidative stress, apoptosis, and inflammation. These bacteria-induced changes can impair semen parameters and subsequently cause infertility. Given the significant destructive effect of some bacteria on sperm function and male fertility, in this study, we reviewed the impact of male urogenital bacteria on spermatogenesis and sperm functions as well as the underlying mechanisms by which the bacteria can damage sperm.

Optimization of Porcine Ovarian Follicle Isolation Methods for Better Developmental Potential.

In the present study, we present a comparative analysis among the outputs of porcine follicle isolation using either mechanical technique alone or in combination with enzymes, proposing an optimized protocol useful for all further applications related to follicle in vitro growth and reproductive tissue engineering. The porcine follicles were isolated using mechanical technique alone (hand blender and scalpels) or in combination with collagenase or Liberase Dispase High (DH) at different doses applying different protocols. Finally, the number, morphology, and stage of isolated follicles were compared between the protocols. Moreover, the follicle viability (live/dead assay) and morphology (rhodamine phalloidin and 4',6-diamidino-2-phenylindole staining and scanning electron microscopy analysis) were evaluated after 10 days of culture. We found an optimum protocol for intact follicle isolation using the mechanical technique in combination with enzymes at a concentration of 0.5 mg/mL. However, the number of total isolated follicles and primordial follicles was significantly higher when collagenase was used compared to Liberase DH (p < 0.05), while Liberase DH could isolate a significantly higher percentage of preantral follicles. After 10 days of culture, the morphology and health status of follicles were statistically higher when Liberase DH was used in comparison with collagenase. Moreover, on the follicles extracted with Liberase DH, it was possible to observe theca cells covering part of the follicle surface. In conclusion, we demonstrated that the intact primary or secondary follicles could not be obtained using only mechanical methods, which led to the isolation of denuded oocytes and dramatically damaged follicles. We concluded that the collagenase-based follicle isolation could negatively affect the morphology and developmental potential of the follicles. Moreover, the incubation of ovarian cortex tissues with Liberase DH solution is an optimized protocol for porcine ovarian follicle isolation with developmental competence. Impact statement Isolation and in vitro maturation of follicles can pave the way for activities on reproductive tissue engineering (REPROTEN) and developing an artificial ovary. In this regard, the standardization and optimization of the extraction methods are pivotal for the design of experiment of follicle in vitro growth. In the present study, we provided a comparative analysis among the outputs of porcine follicle isolation using either mechanical technique alone or in combination with collagenase or Liberase DH, proposing an optimized protocol useful for all further applications related to follicles' in vitro growth and REPROTEN.