TUNEL labeling with BrdUTP/anti-BrdUTP greatly underestimates the level of sperm DNA fragmentation in semen evaluation.

Many studies have now confirmed that sperm DNA fragmentation (SDF) is associated with a poorer outcome of some forms of assisted reproduction technology. For this reason, SDF is an important parameter to evaluate in male fertility assessment. TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) assay coupled to flow cytometry is one of the most promising methods for SDF quantification. Several kits for the detection of DNA fragmentation are currently available on the market and all are recommended as equally appropriate to quantify SDF. In this work we compared for the first time the efficacy of two different types of TUNEL kits for SDF quantification: one using an indirect antibody-based labeling system (BrdUTP/fluorescein-anti-BrdUTP) and another using a direct labeling system (fluorescein-dUTP). We demonstrated that TUNEL indirect labeling system largely underestimates SDF when compared with the direct labeling, the differences ranging from 19.2% to 85.3% (p<0.05, n = 22). We observed that these differences were most pronounced among dead spermatozoa where indirect labeling stained 40.1% [23.6%, 58.2%] and the direct system 65.7% [36.5%, 90.9%] (n = 10, p<0.05). Interestingly, we found that both systems stained the living spermatozoa with the same efficiency. We showed that the differences are due to the steric hindrance of the antibody during its binding to the BrdUTP. Indeed, after sperm DNA decondensation, the percentages of TUNEL positivity increased significantly from 46.3% [31.8%, 61.7%] to 97.5% [96.1%, 98.8%] (p<0.05, n = 5). Our results are important for future use of TUNEL in clinical practice. Laboratories relying on the use of an antibody-based system heavily underestimate SDF, most particularly in infertile patients with reduced sperm motility. As a consequence, the kit using BrdUTP/fluorescein-anti-BrdUTP should not be recommended as a method to assay DNA damage in semen. This study represents one further step in the standardization of TUNEL among laboratories.

Endometrial thickness influences neonatal birth weight in pregnancies with obstetric complications achieved after fresh IVF-ICSI cycles.

PURPOSE: Pregnancy-associated complications, duration of gestation and parity are well-known predictors of neonatal birth weight. Assisted reproductive technology (ART) affects neonatal birth weight as well. Endometrial thickness as measured on the day of HCG triggering may therefore impact on the neonatal birth weight. METHODS: The data of 764 singleton deliveries achieved after fresh transfer between November 1997 and 2014 were collected retrospectively with the intention to analyze the relationship of maternal and neonatal characteristics with endometrial thickness and the possible predictive value of endometrial thickness on neonatal birth weight. RESULTS: Higher maternal age (p < 0.001), diminished ovarian reserve (p < 0.001), endometriosis (p = 0.008) and hypogonadotropic hypogonadism (p < 0.001) predicted thin endometrium. Neonatal birth weight (p = 0.004), longer duration of pregnancy (p = 0.008), parity (p = 0.026) and higher maternal BMI (p = 0.003) were correlated significantly with the degree of endometrial proliferation. Endometrial thickness strongly predicted neonatal birth weight (p = 0.004). After adjusting regression analysis for maternal age and BMI, parity, neonatal gender and pregnancy duration, endometrial thickness remained predictive for neonatal birth weight in pregnancies with obstetric complications (p = 0.017). In uneventful pregnancies duration and parity are determinants of neonatal birth weight. CONCLUSIONS: Our findings suggest that endometrial thickness is an additional ART-related factor influencing neonatal birth weight. This finding should be confirmed in large cohort studies.

Gene expression profiles of similarly derived human embryonic stem cell lines correlate with their distinct propensity to exit stemness and their different differentiation behavior in culture.

Four normal-karyotype human embryonic stem cell (hESC) lines were generated using the same protocol and maintained under identical conditions. Despite these precautions, gene expression patterns were found to be dissimilar among the four lines. The observed differences were typical of each cell line, correlated with their distinct propensity to exit stemness, created heterogeneity among the cells during cell line maintenance, and correlated with their altered capacity as a source of differentiated cells. The capacity of some cell lines to give rise to more, and more mature, neurons within comparable time frames of directed differentiation reflected the distinct proportions of cells already predifferentiated at the onset. These findings demonstrate that the subsequent stages of neural differentiation were altered both in a quantitative and timely fashion. As a consequence, cell lines with apparent better and quicker ability to produce neurons were actually the less capable of reproducing proper differentiation. Previous data suggested that cell lines able to generate more neurons faster would be more suitable to clinical application. Our analysis of the differentiation process strongly suggests the opposite. The spontaneous tendency to predifferentiate of any particular hESC line should be known because it clearly impacts further experimental results.

Isolation of spermatozoa with low levels of fragmented DNA with the use of flow cytometry and sorting.

OBJECTIVE: To develop a method to select spermatozoa with low DNA fragmentation rates for assisted reproduction technologies (ART). DESIGN: Multistep prospective cohort study. SETTING: University hospital. PATIENT(S): Semen samples of 34 infertile men were prepared in parallel with swim-up and fluorescence-activated cell sorting (FACS), and 11 semen samples were used for testing the staining strategy. INTERVENTION(S): Flow cytometric sorting of YO-PRO-stained spermatozoa. MAIN OUTCOME MEASURE(S): Assessment of recovery of spermatozoa and purity after sorting, quantification of sperm DNA fragmentation and viability after sorting and after swim up preparation. RESULT(S): Staining with YO-PRO could be performed successfully in regular culture medium, both dead and apoptotic spermatozoa were labeled without the dye entering the viable spermatozoa. Compared with the conventional swim-up method, the sorted viable population showed a significantly reduced number of spermatozoa with fragmented DNA. CONCLUSION(S): A novel method has been developed, which not only might improve the outcome of ART, but can also help to clarify the ongoing controversy of the role of DNA fragmentation in male infertility.

First successful pregnancy in Switzerland after prospective sex determination of the embryo through the separation of X-chromosome bearing spermatozoa.

QUESTION UNDER STUDY: The feasibility and the potential advantages of separating X-chromosome bearing spermatozoa for the prevention of a severe X-chromosome linked disorder with the use of intracytoplasmic sperm injection are presented. METHOD: A carrier of muscular dystrophy type Becker was treated with intracytoplasmic sperm injection, using spermatozoa previously stained with the Hoechst dye 33342 and sorted with flow cytometry. RESULTS: After transfer of one single blastocyst, an intrauterine pregnancy arose. In the ninth week of gestation, the female sex of the embryo was confirmed with proof of absence of the SRY gene of the Y-chromosome. After normal pregnancy, the patient delivered a healthy daughter. CONCLUSIONS: The staining of spermatozoa with specific markers and sorting with flow cytometry provides a means of preventing significant disease in the offspring and may help in reducing the number of surplus embryos needed for preimplantation genetic diagnosis.

[Present-day reproductive medicine and future possibilities]. / Die Reproduktionsmedizin heute und Chancen für die Zukunft.

Despite the controversial discussions, which have accompanied it since its first introduction of into clinical medicine particularly in Switzerland, assisted reproductive medicine has come to acquire a firm standing in present-day society. This broad acceptance is the logical consequence of the availability of efficient methods of family planning which started with the introduction of oral contraception in the early sixties. Over time assisted reproductive medicine has grown with a considerable degree of efficacy due to improvements in the culture conditions in the embryology laboratory and due to sophisticated ovarian hyperstimulation. In conjunction with steadily rising treatment numbers the latter has contributed to a high incidence of multiple deliveries, which now is considered a complication of assisted reproduction and current opinion demands that multiple pregnancies must be avoided as much as possible. The experience in various countries has demonstrated that through the selection of one single embryo with high developmental capacity the incidence of multiple gestations can be lowered to virtually none. However, the restrictive legislations in vigour both in Switzerland and in Germany impede such an endeavour. Still, the significantly higher pregnancy rates achieved by extending the culture of embryos up to the blastocyst stage clearly demonstrate that embryology holds the clues to further considerable improvements of assisted reproductive technology. Through the rapid development of three-dimensional culture techniques, immature ovarian follicles e.g. primordial follicles may be cultured up to the mature Graafian stage thereby avoiding the need to stimulate the ovaries.

The multipotency of luteinizing granulosa cells collected from mature ovarian follicles.

Graafian ovarian follicles consist of follicular fluid, one single mature oocyte, and several hundred thousands of granulosa cells (GCs). Until now, luteinizing GCs have been considered to be terminally differentiated, destined to undergo death after ovulation. Present concepts of luteal function, endocrine regulation of early pregnancy, and the recruitment of new ovarian follicles are all based on the cyclical renewal of the entire population of GCs. We now demonstrate that luteinizing GCs isolated from the ovarian follicles of infertile patients and sorted with flow cytometry based upon the presence of their specific marker, the follicle-stimulating hormone receptor (FSHR), can be maintained in culture over prolonged periods of time in the presence of the leukemia-inhibiting factor (LIF). Under those conditions the markers of GC function such as FSHR and aromatase gradually disappeared. POU5F1 (POU domain, class 5, homeobox 1), a typical stem cell marker, was expressed throughout the culture, but germ line cell markers such as nanog, vasa, and stellar were not. Mesenchymal lineage markers such as CD29, CD44, CD90, CD105, CD117, and CD166, but not CD73, were expressed by substantial subpopulations of GCs. The multipotency of a subset of GCs was established by in vitro differentiation into other cell types, otherwise not present within ovarian follicles, such as neurons, chondrocytes, and osteoblasts. Follicle-derived stem cells were also able to survive when transplanted into the backs of immunoincompetent mice, in vivo generating tissues of mesenchymal origin. The unexpected findings of multipotency of cells with prolonged lifespans originating from ovarian follicles are likely to have a significant impact on evolving theories in ovarian pathophysiology, particularly with reference to ovarian endometriosis and ovarian cancer.

First delivery of healthy offspring after freezing and thawing of oocytes in Switzerland.

The interest in long-term storage of uninseminated oocytes through cryopreservation has seen a recent upsurge, because it provides the potential to assist young women to postpone childbirth after having overcome a malignant disease or delaying childbirth until after management of a professional career. The low fertilisation rate of frozen/thawed oocytes in earlier feasibility trials can now be improved by using intracytoplasmic sperm injection (ICSI) for assisting the penetration of the spermatozoon through the oocyte's hardened zona pellucida. Another reason for the reported low success rates of oocyte cryopreservation in earlier studies may have been the low developmental potential of spare oocytes, which were available for experimental cryopreservation. Oocytes retrieved from supernumerary follicles in women treated with gonadotropins for ovulation induction and intrauterine insemination can be used for the optimisation of cryostorage of uninseminated oocytes. We intended to investigate to what extent the well-established and successful cryopreservation protocols for pronucleate oocytes are also applicable for the cryopreservation of uninseminated oocytes. We herewith report the first successful pregnancy and delivery of frozen/thawed oocytes in Switzerland, which were inseminated with ICSI. In unbiased treatment groups the freezing and thawing of uninseminated oocytes and pronucleate oocytes give comparable results, if the additional manipulation during ICSI was taken into account.

Elaboration of a working model for the involvement of inhibin A as a mediator of the preovulatory rise of progesterone levels.

During the final days of follicular development, exogenously administered follicle-stimulating hormone (FSH) produces a rise in serum progesterone level. The aim of the present study was to investigate the possible source and regulation of this preovulatory progesterone surge. Four sets of matching treatments with gonadotropins for in vitro fertilization and intracytoplasmic sperm injection were selected from a cohort of 953 treatments in 244 couples. Half of these four sets of treatments were selected based on the unusual course of the progesterone concentration during follicular development. The first set of 11 cycles with early termination of gonadotropin administration for prolonged coasting were compared with a set of 12 cycles with similar estradiol levels but with uninterrupted ovarian stimulation. Another set of 12 cycles with low preovulatory progesterone levels (<2 nmol/l) were matched with ten cycles with normal preovulatory progesterone levels (>2 nmol/l). The sera of these four selected sets of treatments were stored for subsequent measurement of the concentrations of inhibin A, inhibin B, activin A and leptin. During ovarian hyperstimulation serum levels of inhibin A correlated significantly with those of progesterone (p < 0.001), whereas this correlation disappeared after the withdrawal of FSH administration. The rapid fall of progesterone levels during prolonged coasting contrasts with the continuing rise of estradiol concentration and indicates that the theca interna, not the granulosa, is the major source of preovulatory progesterone. Women failing to produce any increment of progesterone levels at the end of follicular development had significantly lower levels of inhibin A (p < 0.05), indicating that inhibin A may well be involved in mediating the signal of FSH from the granulosa to the theca interna.

Apoptosis and differentiation induced by staurosporine in granulosa tumor cells is coupled with activation of JNK and suppression of p38 MAPK.

We report here that staurosporine can induce apoptosis or differentiation of granulosa tumor cells depending on its dosage. In presence of staurosporine concentrations > 50 nM, apoptosis was triggered in human granulosa cell tumor cells COV434. In the presence of concentrations < 50 nM, the shape of the otherwise globular granulosa cells differentiated into a flattened epithelioid-like appearance. The process was associated by the induction of prostaglandin synthase-2 (PGS-2) and C/EBPbeta expression and by an increase in progesterone production in the supernatant culture medium. The observed effects of staurosporine were synergized by forskolin. With phosphorylation-specific Western blotting and protein kinase assays, it was demonstrated that staurosporine suppresses the phosphorylation of p38 and activates JNK. These results suggest that p38MAPK and JNK signal transduction pathways were involved in the regulation of granulosa cell differentiation by staurosporine. These results may indicate the usefulness of staurosporine or its analogs for the development of a future medical treatment of granulosa tumors.

Progesterone serum levels during the follicular phase of the menstrual cycle originate from the crosstalk between the ovaries and the adrenal cortex.

BACKGROUND: The preovulatory rise of progesterone is important for ovulation, but both its regulation and its origin are controversial. Three experiments were performed to determine whether follicular phase progesterone arises from the ovary, the adrenal cortex or both. METHODS: The first study was performed in patients scheduled for assisted reproduction, who received a long-acting GnRH agonist either during intake of an oral contraceptive or during the luteal phase of an otherwise untreated menstrual cycle. The second study was also performed during down-regulation with a GnRH agonist: some patients with elevated progesterone levels received dexamethasone (DXM). Others with similarly elevated basal progesterone levels and those with low progesterone levels were not treated with DXM and served as controls. Finally, adrenocorticotrophic hormone (ACTH) tests were performed in normocyclic volunteers both during early and late follicular phase and during intake of a contraceptive pill. RESULTS: During the suppression of endogenous gonadotrophin secretion progesterone levels rose after the administration of ACTH, but not of GnRH. DXM did not prevent the preovulatory rise of the serum progesterone concentration. The ACTH-stimulated concentration of progesterone and of 17alpha-hydroxyprogesterone were significantly reduced during intake of ethinyl estradiol. CONCLUSIONS: Progesterone arises in the adrenal cortex during most of the follicular phase, whereby its function is modulated by an unknown ovarian factor, which is suppressed by ethinyl estradiol. The source of progesterone shifts towards the ovaries prior to ovulation.