Ozgur Cinar

Biology of stem cells in human umbilical cord stroma: in situ and in vitro surveys.

Cells in the umbilical cord stroma have gained attention in recent years; however, differentiation to certain lineages in humans has been demonstrated in few studies. Unlike bone marrow MSCs, human umbilical cord stroma cells (HUCSCs) are far from being well characterized. This study attempts to describe proliferation, structural, and differentiation properties of these cells to account for their exceptional nature in many aspects. Cellular dynamics, cellular structure, and the degree of transformations during expansion and differentiation into mesenchymal and neuronal lineages were examined in vitro over a 10‐month period. Comparisons with human bone marrow MSCs regarding differentiation were performed. HUCSCs in culture revealed two distinct cell populations, type 1 and type 2 cells, that possessed differential vimentin and cytokeratin filaments. Corresponding cells were encountered in cord sections displaying region‐specific localization. α‐Smooth muscle actin and desmin filaments, which were evident in cord sections, diminished through passages. No difference was noted regarding type 1 and type 2 cells in differentiation to chondrogenic, adipogenic, and osteogenic lineages, whereas a preferential differentiation was noted in neuronal lineage. Relative success was achieved by production of chondrocytic spheres and osteogenic monolayers, whereas adipocytes were immature compared with bone marrow MSCs. The presence of neuronal markers suggests that they transform into a certain state of maturity under neurogenic induction. Conclusively, HUCSCs retain their original phenotype in culture without spontaneous differentiation, have a limited lifespan, and bear multipotent stem cell characteristics. Given these characteristics, they may be generally considered progenitor cells if manipulated under appropriate conditions and deserve further study to be potentially used in cell‐based therapies.

Factors affecting pregnancy outcome of intrauterine insemination cycles in couples with favourable female characteristics

Summary The aim of the presented study is to determine the effect of different sperm parameters on the pregnancy rate of intrauterine insemination (IUI) cycles in women with favourable fertility characteristics treated for infertility. Medical records of 212 infertile couples who had undergone a total of 253 cycles were reviewed retrospectively. Inclusion criteria for women were age <35 years, antral follicle count >5, FSH <15 IU/ml, and at least one patent tube documented by HSG or laparoscopy. Clinical pregnancy rates were achieved as 15.8% per cycle, and 18.8% per couple. Woman’s age, partner’s age, total number of motile sperm (TMS) and motility, significantly influenced pregnancy rate. Pregnancy rate was the highest when women were aged <25 and TMS >10 × 106. Partner’s age significantly affected the pregnancy rate per cycle in women aged <30 years and TMS >10 × 106. Woman’s age (OR: 5.4 95% CI: 1.2–24.3) and TMS (OR: 0.06 95% CI: 0.003–0.89) were predictor variables as regards to pregnancy. Pregnancy rate was the highest in IUI cycles when woman was <25 years old, TMS was >10 × 106, and morphology was >4%. Male age was found to be another determining factor for IUI success, even if they had a normal spermiogram.

Does cigarette smoking really have detrimental effects on outcomes of IVF

OBJECTIVE Cigarette smoke contains many toxic chemicals associated with poor reproductive outcome and reduced fertility. It also has a negative effect on sperm motility and morphology. The aim of this study was to analyze the effects of male and female cigarette smoking on the outcomes of in vitro fertilization (IVF). STUDY DESIGN In this comparative prospective analysis, the effects of smoking on outcomes of IVF including semen parameters, oocyte quality, fertilization rate, transfer day embryo scores and pregnancy rates were analyzed. For this purpose, patients were grouped based on their follicular or seminal fluid cotinine (a nicotine metabolite) levels as smokers and non-smokers (non-smokers: female (n=171), male (n=118), smokers: female (n=43), male (n=96)). RESULTS The mean age and baseline hormonal levels of all groups were found to be comparable. While the numbers of total and mature oocytes collected were higher in the smoker group (p=0.005 and p=0.006, respectively), oocyte quality index, fertilization rate, embryo development rate and pregnancy rate were not significantly different between the groups (p>0.05). Analysis based on the type of ovarian hyperstimulation protocol (GnRH agonist, antagonist and others) showed that within the antagonist group the mean age of smokers was significantly lower and the total number of collected oocytes was significantly higher compared with non-smokers. Cigarette smoking among men did not have a significant negative effect on outcomes of IVF whether their partners were smokers or nonsmokers. Regression analysis did not give any significant difference when male and/or female smoking status was analyzed for fertilization rates, transferred embryo qualities and clinical pregnancy rates. CONCLUSION Cigarette smoking does not have detrimental effects on outcomes of IVF.

The relationship between meiotic spindle imaging and outcome of intracytoplasmic sperm injection: a retrospective study

Meiotic spindle analysis with a non-invasive technique, the PolScope, is used to protect the meiotic spindle from damage during microinjection. To evaluate the predictive feature of PolScope, we have designed a retrospective study to analyse the correlation between the meiotic spindle visualisation with regard to spindle location and outcomes of assisted reproductive technologies (ART), including patient age, previous cycles, the number of the collected oocytes, fertilisation rates (FR), pronuclear scoring (PNS) and embryo scoring of the days from two to five. All of the data belonging to 1496 oocytes from 190 patients were statistically analysed. We found that the oocytes having PolScope visualised spindle have higher FR, and also observed that when the spindle located at 0°–30° according to the first polar body, gave the highest FR. PNS gave higher scores in the spindle visualised group, but spindle angle did not affect PNS outcomes. Although a correlation was found between spindle visualisation and developed embryo qualities, particularly at day 2 and 3, spindle angles did not affect embryo quality. We conclude that PolScope microscopy has an efficiency to estimate FR, and cleavage stage embryo development.

Can serum oestradiol be a predictor of quality of oocytes and embryos, maturation of oocytes and pregnancy rate in ICSI cycles?

Our aim was to assess the influence of ratios of oestradiol (E2) to either number of follicles ≥14 mm on the day of human chorionic gonadotropin administration (E2/fol) or the number of oocytes retrieved (E2/o) during oocyte pick up and total serum E2 levels on the day of embryo transfer (ETE2) on the outcome of ICSI cycles. The assessed outcomes were number of oocytes retrieved (NRO), number of mature oocytes (NMO), number of fertilised oocytes (NFO), number of transferred embryos (NTE), qualities of oocytes (OQS), qualities of embryos (EQS) and pregnancy rates (PR). Two hundred and twenty-seven ICSI-ET cycles admitted to our IVF clinic during a 2-year period with normal ovarian reserve receiving long luteal GnRH agonist protocol were included. The E2/fol levels correlated positively with NRO (r = 0.202, p = 0.002), NMO (r = 0.199, p = 0.003) and NFO (r = 0.159, p = 0.018). However, we observed negative correlations between E2/o and NMO (r = −0.329, p <0.001), NFO (r = −0.219, p = 0.001), EQ5 (r = −0.203, p = 0.040). Oocyte quality scores were not affected from either E2/fol or E2/o levels. Implantation, clinical and ongoing PRs were comparable between groups categorised due to E2/fol, E2/o and ETE2. It seems that high E2/fol ratio may have beneficial effects on NRO, NMO and NFO while E2/o may adversely affect these parameters. Neither of the E2 levels is associated with pregnancy rates in women with normal ovarian reserve.

Potential role of decidual apoptosis in the pathogenesis of miscarriages

To investigate the existence and the distribution of decidual apoptosis in normal pregnancies and miscarriages (spontaneous and recurrent), a comparative immunofluorescent tissue labelling of normal control (n = 12) and miscarried pregnancies (n = 24) was designed. Evaluation of the existence and distribution of decidual apoptosis in normal pregnancies and miscarriages, characterization of the apoptotic cell types and the involvement of caspase-dependent pathways was analyzed with TUNEL, anti-active caspase-3, anti-pancytokeratin and anti-CD45 antibodies. Normal decidua showed few apoptotic cells, whereas decidua from recurrent miscarriages had a significantly higher number of apoptotic cells preferentially localized to the sub-epithelial and periarteriolar regions, where the onset of decidualization occurs. Apoptosis occurred via a caspase-dependent pathway. Neither immune nor epithelial cells were positively stained for any apoptotic markers. The increased number of apoptotic cells, which are strictly restricted to the periarteriolar stroma particularly in recurrent miscarriages leads us to suggest that decidual apoptosis could result a series of cellular dysfunctions that may threaten the course of pregnancy.

Ultrastructural alterations in human decidua in miscarriages compared to normal pregnancy decidua.

Background. Pregnant endometrial stroma, an immunologically privileged site in the female reproductive system, is enriched by decidual and natural killer (NK) cells. Since the cellular microenvironment in early pregnancy from the decidual tissues of normal and miscarriage cases has gained importance, with special emphasis on cell‐to‐cell contacts, we aimed to document the plastic structure of the cellular milieu in normal and miscarriage decidua. Methods. Endometrial biopsies were obtained from women after legal curettage or women who had been treated by curettage after miscarriage. Samples were analysed in a light microscope (LM), a scanning electron microscope (SEM) and a transmission electron microscope (TEM). Results. Decidual cells possess several polyploidic protrusions on cell membranes. NK cells were distributed among decidual cells. Decidual cells were found to develop gap junctions in the interfaces between each other. Their cytoplasms were also found to possess well‐developed protein synthesising organelles. Decidual cells obtained from miscarriages showed a moderate degree of degeneration and, in between, a decreased number of junctional complexes. Mononuclear cell infiltration was found to be significantly low. Conclusion. We conclude that decidual cells during early pregnancy build a series of miniature cell‐cell contacts to assemble a proper endometrial milieu. In contrast, in miscarriage samples, those intercellular communications seem lacking, associated with an increased number of NK cells, a phenomenon which obviously alters proper implantation and leads to the induction of embryonic disgenesis and miscarriage.

Does combining magnetic-activated cell sorting with density gradient or swim-up improve sperm selection?

PurposeThe present study aimed to evaluate whether combining the magnetic-activated cell sorting (MACS) with density-gradient (DG) or swim-up (SU) sperm separation techniques can improve sperm selection to obtain higher quality spermatozoa.MethodsTwo commonly used sperm selection techniques, SU and DG, were compared to MACS combined with either SU or DG. Spermatozoa obtained from normozoospermic (n = 10) and oligozoospermic (n = 10) cases were grouped as SU, DG, SU+MACS, and DG+MACS followed by the analysis of sperm morphology, motility, DNA integrity, and the levels of Izumo-1 and PLCZ proteins.ResultsAlthough spermatozoa obtained by SU or DG when combined with MACS have improved aspects when compared to SU or DG alone, results did not reach a statistically significant level. Moreover, separation with MACS caused a significant loss in the numbers of total and rapid progressive spermatozoa.ConclusionsConsidering the cost/benefit ratio, MACS application together with traditional techniques may only be preferred in certain cases having higher concentrations of spermatozoa, but it does not seem to be an ideal and practical sperm selection technique for routine use.

Umbilical cord mesenchymal stromal cell transplantations: A systemic analysis of clinical trials

The advances and success of umbilical cord-derived mesenchymal stromal cells (UC-MSCs) in experimental disease animal models have fueled the development of targeted therapies in humans. The therapeutic potential of allogeneic transplantation of UC-MSCs has been under examination since 2009. The purpose of this systematic analysis was to review the published results, limitations and obstacles for UC-MSC transplantation. An extensive search strategy was applied to the published literature, 93 peer-reviewed full-text articles and abstracts were found published by early August 2017 that investigated the safety, efficacy and feasibility of UC-MSCs in 2001 patients with 53 distinct pathologies including many systemic/local, acute/chronic conditions. Few data were extracted from the abstracts and/or Chinese-written articles (n = 7, 8%). Importantly, no long-term adverse effects, tumor formation or cell rejection were reported. All studies noted certain degrees of therapeutic benefit as evidenced by clinical symptoms and/or laboratory findings. Thirty-seven percent (n = 34) of studies were found published as a single case (n = 10; 11%) or 2-10 case reports (n = 24; 26%) with no control group. Due to the nature of many stem cell-based studies, the majority of patients also received conventional therapy regimens, which obscured the pure efficacy of the cells transplanted. Randomized, blind, phase 1/2 trials with control groups (placebo-controlled) showed more plausible results. Given that most UC-MSC trials are early phase, the internationally recognized cell isolation and preparation standards should be extended to future phase 2/3 trials to reach more convincing conclusions regarding the safety and efficacy of UC-MSC therapies.

Estradiol supplementation in intracytoplasmic sperm injection cycles with thin endometrium

Objective: To evaluate the efficacy of estradiol supplementation starting on the day of human chorionic gonadotrophin (hCG) in patients with thin endometrium in intracytoplasmic sperm injection (ICSI) cycles. Methods: A total of 117 consecutive patients with the endometrial thickness on the hCG day ≤8 mm were rewieved. Estradiol supplementation was given in 57 patients and the remaining 60 patients were accepted as control group. Estradiol supplemented (ES) group received estradiol hemihydrate 4 mg/day started on the day of hCG. Luteal phase was supported using the vaginal progesterone gel in both groups. Clinical pregnancy rate, implantation rate, miscarriage rate, endometrial thickness on the day of oocyte pick-up and on the day of embryo transferred were accepted as main outcome measures. Results: There were no statistical differences in terms of clinical pregnancy rate (28.1% vs. 23.3%), implantation rate (16% vs. 10.4%), miscarriage rate (21% vs. 31.6%), endometrial thickness on the oocyte pick-up day (8.5 ± 1.8 vs. 8.4 ± 1.4, mm) and embryo transferred day (9.6 ± 2.9 vs. 10.3 ± 2.4, mm) in the ES group vs. control group. Conclusion: Estradiol supplementation starting on the hCG day for the patients with thin endometrium does not provide any benefit on the pregnancy outcome in ICSI cycles.