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The central thesis regarding the human ovaries is that, although primordial germ cells in embryonal ovaries are of extraovarian origin, those generated during the fetal period and in postnatal life are derived from the ovarian surface epithelium (OSE) bipotent cells. With the assistance of immune system-related cells, secondary germ cells and primitive granulosa cells originate from OSE stem cells in the fetal and adult human gonads. Fetal primary follicles are formed during the second trimester of intrauterine life, prior to the end of immune adaptation, possibly to be recognized as self-structures and renewed later. With the onset of menarche, a periodical oocyte and follicular renewal emerges to replace aging primary follicles and ensure that fresh eggs for healthy babies are always available during the prime reproductive period. The periodical follicular renewal ceases between 35 and 40 yr of age, and the remaining primary follicles are utilized during the premenopausal period until exhausted. However, the persisting oocytes accumulate genetic alterations and may become unsuitable for ovulation and fertilization. The human OSE stem cells preserve the character of embryonic stem cells, and they may produce distinct cell types, including new eggs in vitro, particularly when derived from patients with premature ovarian failure or aging and postmenopausal ovaries. Our observations also indicate that there are substantial differences in follicular renewal between adult human and rat ovaries. As part of this chapter, we present in detail protocols utilized to analyze oogenesis in humans and to study interspecies differences when compared to the ovaries of rat females.

The purpose of this prospective randomized study was to evaluate use of a hyaluronan-rich transfer medium in fresh and frozen–thawed single blastocyst transfer. The study included 279 single blastocyst transfers in women aged

Irma Virant-Klun Department of Obstetrics and Gynaecology, University Medical Center Ljubljana, Ljubljana, Slovenia Abstract: In spite of generally accepted dogma that the total number of follicles and oocytes is established in human ovaries during the fetal period of life rather than forming de novo in adult ovaries, some new evidence in the field challenges this understanding. Several studies have shown that different populations of stem cells, such as germinal stem cells and small round stem cells with diameters of 2 to 4 µm, that resembled very small embryonic-like stem cells and expressed several genes related to primordial germ cells, pluripotency, and germinal lineage are present in adult human ovaries and originate in ovarian surface epithelium. These small stem cells were pushed into the germinal direction of development and formed primitive oocyte-like cells in vitro. Moreover, oocyte-like cells were also formed in vitro from embryonic stem cells and induced pluripotent stem cells. This indicates that postnatal oogenesis is not excluded. It is further supported by the occurrence of mesenchymal stem cells that can restore the function of sterilized ovaries and lead to the formation of new follicles and oocytes in animal models. Both oogenesis in vitro and transplantation of stem cell-derived “oocytes” into the ovarian niche to direct their natural maturation represent a big challenge for reproductive biomedicine in the treatment of female infertility in the future and needs to be explored and interpreted with caution, but it is still very important for clinical practice in the field of reproductive medicine. Keywords: human, follicle, oocyte, stem cells

An adequate analytical method for determination of amino acids can provide a better insight in the metabolism of in vitro human embryo cultures, increasing the success rate of embryo implantation. Since individual amino acid amounts per embryo occur in the nanogram range, GC was the technique of choice, due to its inherent sensitivity and high sample throughput. Amino acids were analyzed as alkyl formate derivatives. The limits of detection (LOD) of all amino acids involved were in the sub-nmol range. The high risk of sample contamination proved to be the major analytical issue, but it could be overcome. For an extended method sensitivity, a simple preconcentration step could also be used.

Surface cells in adult ovaries represent germ line-competent embryonic stem cells. They are a novel type of totipotent progenitors for distinct cell types including female germ cells/oocytes, with the potential for use in the autologous treatment of ovarian infertility and stem cell therapy. Ovarian infertility and stem cell therapy are complex scientific, therapeutic, and socioeconomic issues, which are accompanied by legal restrictions in many developed countries. We have described the differentiation of distinct cell types and the production of new eggs in cultures derived from adult human ovaries. The possibility of producing new eggs from ovarian surface epithelium representing totipotent stem cells supports new opportunities for the treatment of premature ovarian failure, whether idiopathic or after cytostatic chemotherapy treatment, as well as infertility associated with aged primary follicles, and infertility after natural menopause. The stem cells derived from adult human ovaries can also be used for stem cell research and to direct autologous stem cell therapy. This chapter describes general considerations regarding the egg origin from somatic progenitor cells, oogenesis and follicle formation in fetal and adult human ovaries (follicular renewal), including the promotional role of the immune system-related cells in vivo, and possible causes of ovarian infertility. It then provides detailed protocols for the separation and cultivation of adult ovarian stem cells.

The aim was to establish the impact of human granulosa cell apoptosis and reactive oxygen species (ROS) production on fertilization competence of the oocyte, embryo developmental stage and implantation rate.Thirty women undergoing IVF-ET for tubal factor infertility were included; GnRH antagonists and gonadotrophins were used for ovarian stimulation. Granulosa cells were isolated from each aspirated follicle using gradient centrifugation. Apoptosis was studied by flow cytometry using annexin V and propidium iodide. ROS production was studied with hydroethidine staining and analyzed by flow cytometry.There were no differences in characteristics of granulosa cells between the follicles with fertilized and non-fertilized oocytes. The analyzed characteristics of granulosa cells in corresponding follicles had no effect on embryo developmental stage on day 5. The percentage of ROS producing granulosa cells was lower in the follicles giving rise to blastocysts that resulted in implantation compared to those that did not (39.9% versus 69.9%, P = 0.031).Apoptosis and ROS production in granulosa cells have no significant impact on fertilization and do not correlate with the development of blastocysts. An increased percentage of ROS producing granulosa cells results in fewer oocytes retrieved and diminishes implantation rate.

The 50-year-old and currently prevailing view that all oocytes in adult human ovaries persist from the fetal period of life is controversial as it clashes with Darwinian evolutionary theory. Studies of oogenesis and follicular renewal in adult human ovaries, and of the role of hormonal signals and third-party cells (tissue macrophages and T cells), could all be helpful in providing better understanding of the causes of ovarian infertility, its prevention and potential therapy. In addition, the authors recently reported differentiation of distinct cell types and the production of new eggs in cultures derived from premenopausal and postmenopausal human ovaries. It is possible that fertilisation of such eggs will open up new opportunities for providing genetically related children to infertile women for whom conventional in vitro fertilisation has failed. As ovarian stem cells appear to represent a new type of totipotent adult stem cell, they could also be utilised for autologous stem cell therapy of degenerative diseases, without any involvement of allogeneic embryonic stem cells and somatic cell nuclear transfer.

Surface cells in adult ovaries represent germ line-competent embryonic stem cells. They are a novel type of totipotent progenitors for distinct cell types including female germ cells/oocytes, with the potential for use in the autologous treatment of ovarian infertility and stem cell therapy. Ovarian infertility and stem cell therapy are complex scientific, therapeutic, and socioeconomic issues, which are accompanied by legal restrictions in many developed countries. We have described the differentiation of distinct cell types and the production of new eggs in cultures derived from adult human ovaries. The possibility of producing new eggs from ovarian surface epithelium representing totipotent stem cells supports new opportunities for the treatment of premature ovarian failure, whether idiopathic or after cytostatic chemotherapy treatment, as well as infertility associated with aged primary follicles, and infertility after natural menopause. The stem cells derived from adult human ovaries can also be used for stem cell research and to direct autologous stem cell therapy. This chapter describes general considerations regarding the egg origin from somatic progenitor cells, oogenesis and follicle formation in fetal and adult human ovaries (follicular renewal), including the promotional role of the immune system-related cells in vivo, and possible causes of ovarian infertility. It then provides detailed protocols for the separation and cultivation of adult ovarian stem cells.

In this study we sought to determine whether intracytoplasmic sperm injection (ICSI) could improve the efficacy of treatment with gonadotrophins in gonadotrophin-deficient men in terms of pregnancy. A series of six adult men (aged 26-47 years) with hypogonadotrophic hypogonadism (HH) is reported: four men with prepubertal isolated idiopathic HH (IIHH) and two adult-onset HH, as part of hypopituitarism secondary to surgical treatment of a pituitary tumour. All were azoospermic. To restore spermatogenesis, all received hormonal treatment with intramuscular human menopausal gonadotrophins (HMG) and human chorionic gonadotrophin (HCG) for 2 to 23 months. High basal serum inhibin B was predictive of rapid and complete recovery of spermatogenesis. In the two adult-onset HH, a natural pregnancy was achieved within 3 months. The four men with IIHH underwent ICSI because of poor sperm quality. ICSI using fresh or frozen-thawed ejaculated spermatozoa was performed after 6-23 months of gonadotrophin treatment. ICSI provided good clinical results in terms of fertilization and embryo quality, and resulted in three pregnancies that ended in three term deliveries. In men with oligozoospermia related to prepubertal IIHH, ICSI shortens the hormonal treatment and enhances the chances of pregnancy.

Summary There is no consensus on whether and how male genital tract inflammation affects sperm fertilizing potential. The aims of this prospective study were to evaluate the elastase-inhibitor complex in seminal plasma (s-EI) level, a marker of male genital tract inflammation, in men undergoing in vitro fertilization (IVF) having no clinical signs of inflammation, and its association with semen characteristics, and the predictive role of s-EI for the outcome of IVF in terms of fertilization, embryo development to the blastocyst stage and pregnancy. The study involved 104 male partners of infertile couples with normal spermiogram undergoing IVF. On the day of oocyte retrieval, spermiogram and s-EI assessment using homogeneous immunoassay were performed. The outcome of IVF according to the s-EI level was assessed in 85 cycles with two or more oocytes. In 67 cycles embryos were cultured to the blastocyst stage. Spearman's and Pearson's correlation tests, chi-square test, and multiple linear and logistic regression analyses were used for statistical evaluation. Increased s-EI level (≥250 μg/L) was detected in semen of 32% of men, and in 24% of men without leucocytospermia. We found an association between the s-EI level and semen leucocytes (r = 0.49, p = 0.004) but not with classical sperm characteristics. No correlation between the s-EI level and fertilization was observed. Increased s-EI levels were associated with a poorer blastocyst development rate (p = 0.03) and a higher number of arrested embryos (p = 0.04). Extended embryo culture to the blastocyst stage shows a negative effect of clinically silent male genital tract inflammation on embryo developmental potential.

Background. Slovene data on iatrogenic multiple pregnancy after IVF/ICSI procedures are presented. The influence of elective transfer of two high-quality embryos on the incidence of iatrogenic multiple pregnancy were evaluated.Methods. Retrospective analysis of the Slovene IVF/ICSI data in the period 1983–2000.Results. In 17436 IVF/ICSI cycles 3068 pregnancies were achieved. Of the 2308 deliveries 369 (16%) were twins and 49 (2%) triplets. Of the 2827 newborns 738 (26%) were twins and 148 (5%) triplets. With elective transfer of two high quality embryos in 1999 and 2000 in two of the three Slovene centres for assisted reproduction, the pregnancy rate per transfer increased (32%), triplet deliveries decreased to 2/492 deliveries (0.4%) (p < 0.000), and twin deliveries remained unchanged 111/492 deliveries (23%).Conclusions. After elective transfer of two high-quality embryos and cryopreservation of surplus high-quality embryos in two of the three Slovene centres in the years 1999 and 2000, the problem of iatrogenic triple and higher rate multiple pregnancies has been successfully resolved, but the twin delivery rate still remains too high. Only by elective transfer of a single high-quality embryo the high multiple pregnancy rate after IVF/ICSI procedures could be avoided.

Background. Because of complications related to multiple pregnancies after in vitro fertilization this pathology represent a real problem. Intracytoplasmic sperm injection (ICSI) may lead to higher risk in developing monozygotic pregnancies. We sought to investigate whether the multiple pregnancies after ICSI differ of those consecutive to classical in vitro fertilization (IVF) and if counseling for transfering one or at most two embryos is efficient in preventing multiple pregnancies.Methods. We analyzed retrospectively data concerning the incidence of multiple pregnancies before and after the transfer of limited number of embryos.Results. In transfering two cleaving-stage embryos (day 2) or blastocysts (day 5), when pregnancy rate is high, the incidence of multiple pregnancies is 25 and 17%. After transfer of one blastocyst the highest the pregnancy rate and the lowest the incidence of multiple pregnancies.Conclusions. The best way to avoid multiple pregnancies consist in transferring one or at most two embryos. The monozygotic twin pregnancy we observed was consecutive to the transfer of frozen-thawed blastocyst.

Background. Twin pregnancy rates following the transfer of two embryos in in vitro fertilization (IVF) treatment are high. Therefore, the aim of this study was to evaluate twin pregnancy rates in regard to the developmental stage of the embryo, patient age, and cause of infertility.Methods. A retrospective analysis involved 1222 IVF procedures. Overall pregnancy rates and twin pregnancy rates following the transfer of two blastocysts or two cleavage-stage embryos were assessed. The blastocyst group was divided into four age subgroups, and into four subgroups by the cause of infertility.Results. After the transfer of two blastocysts or two cleavagestage embryos the overall pregnancy rates (45% vs. 9%) and the twin pregnancy rates (18% vs. 5%) were significantly higher in the blastocyst group. The effects of patient age and the transfer of two blastocysts on the overall pregnancy and twin pregnancy rates were significantly greater in the subgroups of patients aged ≤ 30 years (21%) and 31–34 years (20%) than in older patients (35–38 years = 15.7%; ≥ 39 years = 9%). In the blastocyst group the twin pregnancy rate was significantly higher in patients with tubal (21%) and endocrinological causes of infertility (24.5%) than in patients with endometriosis (7%) and uterine malformations (12.5%).Conclusions. After the transfer of two blastocysts twin pregnancy rates are significantly higher in patients with tubal factor or endocrinological causes of infertility younger than 34 years. In this population of women the risk of twin pregnancy can be avoided by the transfer of a single embryo – most developed blastocyst.

Background. Since in in vitro fertilization two embryos – blastocysts are routinely transferred, the twin pregnancy rates are higher than in the general population. The aim of our study was to evaluate the clinical results after elective single-blastocyst transfer.Methods. Retrospectively we analysed clinical results of 696 in vitro fertilization cycles. The cycles were divided into 37 cycles, in which an elective single blastocyst transfer was performed (group 1), and in 659 cycles, in which an elective double blastocyst transfer was performed (group 2). The pregnancy rates, twin pregnancy rates, and spontaneous abortion rates were compared between the two groups. Additionally we evaluated the effect of the method of in vitro fertilization (classical IVF and ICSI) and female age (< 38 years and > 38 years) on the clinical results after a single or double blastocyst transfer.Results. After an elective single or double blastocyst transfer, there was no statistically significant difference in pregnancy rates (30% vs. 46%); after a single blastocyst transfer there were no twin pregnancies, whereas after a double blastocyst transfer the pregnancy rate was 18%. After a double blastocyst transfer, the method of in vitro fertilization did not affect the twin pregnancy rate, whereas in women younger than 38 years, the twin pregnancy rate was significantly higher than in women aged > 38 years (19% vs. 6%; p < 0,05).Conclusions. Clinical results obtained after an elective single and double blastocyst transfer are comparable. By the transfer of one blastocyst twin pregnancies are avoided. Elective single blastocyst transfer is recommended in women, younger than 38 years.

Background. Due to changes in the socio-economic environment the first pregnancy is being more often postponed to advanced age; an increasing number of women seek medical help for infertility in their late thirties. Degenerative processes in the ovary start as early as after 35 years of age. When the woman is over 38, the signs of hormonal changes occur (FSH, inhibin), the menstrual cycle changes, and fertility is being increasingly reduced. Infertility treatment by assisted reproduction technology (ART) has proved less efficient in older than in younger women.Material and methods. In a retrospective analysis we evaluated the success rates achieved with homologous intrauterine insemination (IUI), in vitro fertilization and embryo-transfer (IVF-ET), intracytoplasmic sperm injection (ICSI), and compared them in regard to the women > 38 years vs. < 38 years.Results. After IUI the pregnancy rate in the women over 38 was 3.7% per patient (1.5% per cycle) and 28.0% (9.9%) in the women younger than 38 years. After IVF-ET the pregnancy rate in the over 38-year group was 16% per patient (14% per cycle), and 28% per patient (25% per cycle) in the less than 38-year group. After ICSI, the pregnancy rate in the group over 38 years was 11% per patient (9% per cycle), and 25% per patient (22% per cycle) in the less than 38-year group. In the analysed population, the spontaneous abortion rate was 26.0% in the group of women aged over 38 years, and 14.0% in the group of women aged less than 38 years.Conclusions. Before introduction of an ART procedure the woman with advanced age should be properly counselled and well informed about poor success of their infertility treatment and high spontaneous abortion rate.

To compare ovarian stimulation with recombinant FSH (rFSH) vs. urinary FSH (uFSH) in terms of hormonal events within ovarian follicles and the outcome of in vitro fertilization.A prospective randomized comparative study of rFSH (n = 70) vs. uFSH (n = 61) ovarian stimulation. Hormone determinations were serum estradiol (E2) on the day of human chorionic gonadotropin (hCG) administration, and E2, androstenedione (A) and testosterone (T) at the time of follicular aspiration in the follicular fluid and serum.The total dose of gonadotropins required and the length of ovarian stimulation were the same in the 2 groups. In follicular fluid the E2 and the A levels were significantly higher in the rFSH group (3,065 +/- 1,646 vs. 2,368 +/- 1,240 nmol/L, P = .004, and 103.7 +/- 51.6 vs. 89.0 +/- 42.3 nmol/L, P = .042, respectively), whereas A:E2 and T:E2 ratios were significantly lower (39.6 +/- 22.5 vs. 52.3 +/- 59.6, P = .042, and 9.1 +/- 4.7 vs. 17.6 +/- 26.9, P = .006, respectively). Serum hormonal levels, number of oocytes retrieved and pregnancy rates did not differ significantly between the groups.rFSH provides results similar to those of uFSH. rFSH enhances steroidogenesis and provokes different androgen/estrogen ratios than does uFSH without influencing the outcome of in vitro fertilization.

The aim of this study was to evaluate the effect of sperm single-stranded DNA, detected by acridine orange (AO), and classical sperm parameters on embryonic quality after ICSI.Before ICSI, the spermatozoa of 183 infertile patients with oligo-, astheno-, teratozoospermia (n = 147), or more than one previous unsuccessful conventional IVF attempt (n = 36) were stained by AO to assess the presence of single-stranded DNA. Two days after ICSI, the embryos of 135 patients were scored for morphology, fragmentation included. Embryos of 48 couples were cultured for 4 days to develop to the morula or blastocyst stage. At most 2 embryos were transferred on Day 2 or 4.When the level of spermatozoa with single-stranded DNA was increased, there was a significantly lower fertilization rate after ICSI. Besides, increased sperm single-stranded DNA resulted in a higher proportion of heavily fragmented embryos on Day 2 (P0.05). In patients with an increased level of spermatozoa with single-stranded DNA, a significantly higher number of embryos were arrested in spite of prolonged culturing (P0.05). Classical sperm parameters did not affect the quality and developmental potential of ICSI-derived embryos. No correlation was found between the level of spermatozoa with single-stranded DNA, pregnancy rate, and live-birth rate achieved by ICSI, except in patients with 0% of spermatozoa with single-stranded DNA, in whom the pregnancy rate was significantly higher.Sperm single-stranded DNA provides additional data on sperm functional capacity in terms of fertilization and embryonic quality after ICSI.

Apoptosis is a widespread phenomena during development. It represents a form of cell death and has a crucial role in tissue homeostasis. Apoptosis is also involved in a number of pathological conditions. Spermatogenesis is a dynamic process of germ cell proliferation and differentiation. During regular spermatogenesis, the number of testicular germ cells degenerate by an apoptotic process. The significance of regulating cell population by apoptosis is more apparent when sperm production is halted. The presence and frequency of apoptosis in germ cells of human testis biopsy specimens were tested. The results confirm the presence of germ cell apoptosis but not the apoptosis of Sertoli cells. The increased apoptotic index was observed in patients with azoospermia in comparison with normal but obstructed spermatogenesis.

Elastase-inhibitor complex was assessed by immunoassay in the seminal plasma of 312 men attending the outpatient infertility clinic. Using receiver operating characteristic (ROC) curve analysis, elastase at the cut-off value of > or =290 ng/ml was shown to be efficient (sensitivity 79.5%, specificity 74.4%) in the detection of genital tract inflammation as defined by leukocytospermia (>1x10(6) leukocytes/ml). The prevalence of increased elastase in 292 infertile men was significantly higher (34%) as compared with that (5%) observed in 20 fertile men (P: = 0.02). Moreover, high elastase concentration (> or =290 ng/ml) was observed in 66 of the 264 men (25%) without leukocytospermia. A significant positive correlation was found between elastase concentration and patient age (r = 0.202, P: < 0.0001) and the number of leukocytes (r = 0.330, P: < 0.0001). A negative correlation was found between elastase concentration and semen volume (r = -0.146, P: = 0.01) and the percentage of spermatozoa with single-stranded DNA (r = -0.194, P: = 0.024), but there was no correlation between elastase and sperm reactive oxygen species production. A higher seminal elastase concentration was significantly associated with tubal damage in female partners (P: < 0.001). After norfloxacine antibiotic therapy, decrease in elastase concentration was observed in 15 (25%) of the 60 treated patients. Tubal damage in the partner negatively affected the response to antibiotic therapy. In conclusion, granulocyte elastase is a reliable screening test for silent genital tract inflammation of the couple. The elastase-inhibitor complex may have a protective effect in reducing sperm DNA denaturation.

In in vitro fertilization (IVF) cycles controlled ovarian hyperstimulation (COH) is established by gonadotropins in combination with gonadotropin-releasing hormone (GnRH) agonists or antagonists, to prevent premature luteinizing hormone (LH) surge. The aim of our study was to improve the understanding of gene expression profile of cumulus cells (CC) in terms of ovarian stimulation protocol and oocyte maturity. We applied Affymetrix gene expression profiling in CC of oocytes at different maturation stages using either GnRH agonists or GnRH antagonists. Two analyses were performed: the first involved CC of immature metaphase I (MI) and mature metaphase II (MII) oocytes where 359 genes were differentially expressed, and the second involved the two GnRH analogues where no differentially expressed genes were observed at the entire transcriptome level. A further analysis of 359 differentially genes was performed, focusing on anti-Müllerian hormone receptor 2 (AMHR2), follicle stimulating hormone receptor (FSHR), vascular endothelial growth factor C (VEGFC) and serine protease inhibitor E2 (SERPINE2). Among other differentially expressed genes we observed a marked number of new genes connected to cell adhesion and neurotransmitters such as dopamine, glycine and γ-Aminobutyric acid (GABA). No differential expression in CC between the two GnRH analogues supports the findings of clinical studies where no significant difference in live birth rates between both GnRH analogues has been proven.

Background The mechanism of aggressive character of ovarian cancer and unsuccessful treatment of women with this deadly disease has been recently explained by the theory of cancer stem cells (CSCs). It has been reported that ovarian carcinogenesis and progression of disease is associated with epithelial-mesenchymal transition (EMT). EMT, a physiological cell process during embryonic development and later in life during regeneration, could, when induced in pathological condition, generate CSCs-like cells. Until now EMT in the ovarian tissue has been mainly studied in cell cultures in vitro. The aim of this study was to focus on in situ morphological changes in the ovarian surface epithelium of tumor tissue in women with epithelial ovarian cancer after we applied the antibodies for markers of EMT vimentin and pluripotency-related markers NANOG, SOX2 and SSEA-4. Methods We analyzed ovarian tissue sections of 20 women with high grade serous ovarian carcinoma. After eosin and hematoxylin staining, used in standard practice, immunohistochemistry was performed for vimentin and markers of pluripotency: NANOG, SSEA-4 and SOX2. We focused on the ovarian surface epithelium in order to observe morphological changes in tumor tissue. Results Among epithelial cells of the ovarian surface epithelium in women with serous ovarian carcinoma we observed a population of small NANOG-positive cells with diameters of up to 5 μm and nuclei, which filled almost the entire cell volumes. These small NANOG-positive cells were in some cases concentrated in the regions with morphologically changed epithelial cells. In these regions, a population of bigger round cells with diameters of 10–15 μm with large nuclei, and positively stained for vimentin, NANOG and other markers of pluripotnecy, were released from the surface epithelium. These cells are proposed as CSCs, and possibly originate from small stem cells among epithelial cells. They formed typical cell clusters, invaded the tissue by changing their round shape into a mesenchymal-like phenotype, and contributed to the manifestation of ovarian cancer. Conclusions Our findings show morphological changes in the ovarian surface epithelium in tumor slides of high grade serous ovarian carcinoma and provide a new population of putative CSCs. Electronic supplementary material The online version of this article (doi:10.1186/s13048-017-0306-7) contains supplementary material, which is available to authorized users.

Background In previous studies it has been found that in cell cultures of human adult ovaries there is a population of small stem cells with diameters of 2–4 μm, which are present mainly in the ovarian surface epithelium and are comparable to very small embryonic-like stem cells (VSELs) from bone marrow. These cells are not observed by histopathologists in the ovarian tissue due to their small size and unknown clinical significance. Because these cells express a degree of pluripotency, they might be involved in the manifestation of ovarian cancer. Therefore we studied the ovarian tissue sections in women with borderline ovarian cancer and serous ovarian carcinoma to perhaps identify the small putative stem cells in situ. Methods In 27 women with borderline ovarian cancer and 20 women with high-grade serous ovarian carcinoma the ovarian tissue sections were stained, per standard practice, with eosin and hematoxylin staining and on NANOG, SSEA-4 and SOX2 markers, related to pluripotency, using immunohistochemistry. We focused on the presence and localization of small putative stem cells with diameters of up to 5 μm and with the nuclei spread over nearly the full cell volume. Results In ovarian sections of both borderline ovarian cancer and serous ovarian carcinoma patients we were able to identify the presence of small round cells complying with the above criteria. Some of these small cells were NANOG-positive, were located among epithelial cells in the ovarian surface epithelium and as a single cell or groups of cells/clusters in typical “chambers”, were found only in the presence of ovarian cancer and not in healthy ovaries and are comparable to those in fetal ovaries. We envision that these small cells could be related to NANOG-positive tumor-like structures and oocyte-like cells in similar “chambers” found in sections of cancerous ovaries, which could support their stemness and pluripotency. Further immunohistochemistry revealed a similar population of SSEA-4 and SOX2-positive cells. Conclusions We may conclude that putative small stem cells expressing markers, related to pluripotency, are present in the ovarian tissue sections of women with borderline ovarian cancer and high-grade serous ovarian carcinoma thus indicating their potential involvement in ovarian cancer. Electronic supplementary material The online version of this article (doi:10.1186/s13048-016-0221-3) contains supplementary material, which is available to authorized users.

Background Sperm of poor quality can negatively affect embryo development to the blastocyst stage. The aim of this comparative prospective randomized study was to evaluate the role of an intracytoplasmic morphologically selected sperm injection (IMSI) in the same infertile couples included in the programme of intracytoplasmic sperm injection (ICSI) due to their indications of male infertility which had resulted in all arrested embryos following a prolonged 5-day culture in previous ICSI cycles. Methods Couples exhibiting poor semen quality and with all arrested embryos following a prolonged 5-day culture in previous ICSI cycles were divided into two groups: Group 1: IMSI group (n = 20) with IMSI performed in a current attempt and Group 2: ICSI group (n = 37) with a conventional ICSI procedure performed in a current attempt of in vitro fertilization. Fertilization rate, embryo development, implantation, pregnancy and abortion rates were compared between current IMSI and conventional ICSI procedures, and with previous ICSI attempts. Results The IMSI group was characterized by a higher number of blastocysts per cycle than the ICSI group (0.80 vs. 0.65) after a prolonged 5-day embryo culture. There was a significantly lower number of cycles with all arrested embryos and cycles with no embryo transfer in the IMSI group versus the ICSI group (0% vs. 27.0%, p = 0.048). After the transfer of embryos at the blastocyst or morula stage (on luteal day 5) a tendency toward higher implantation and pregnancy rates per cycle was achieved in the IMSI group compared to the ICSI group (17.1% vs. 6.8%; 25.0% vs. 8.1%, respectively), although not statistically significant. After IMSI, all pregnancies achieved by the blastocyst transfer were normally on-going, whereas after ICSI, two of three pregnancies ended in spontaneous abortion. After IMSI, two pregnancies were also achieved by the morula stage embryos, whereas after the conventional ICSI procedure, embryos at the morula stage did not implant. Conclusions The IMSI procedure improved embryo development and the laboratory and clinical outcomes of sperm microinjection in the same infertile couples with male infertility and poor embryo development over the previous ICSI attempts.

Introduction The presence of oocytes in the ovarian surface epithelium has already been confirmed in the fetal ovaries. We report the presence of SSEA-4, SOX-2, VASA and ZP2-positive primitive oocyte-like cells in the adult ovarian surface epithelium of a patient with serous papillary adenocarcinoma. Case presentation Ovarian tissue was surgically retrieved from a 67-year old patient. Histological analysis revealed serous papillary adenocarcinoma. A proportion of ovarian cortex sections was deparaffinized and immunohistochemically stained for the expression of markers of pluripotency SSEA-4 and SOX-2 and oocyte-specific markers VASA and ZP2. The analysis confirmed the presence of round, SSEA-4, SOX-2, VASA and ZP2-positive primitive oocyte-like cells in the ovarian surface epithelium. These cells were possibly related to the necrotic malignant tissue. Conclusion Primitive oocyte-like cells present in the adult ovarian surface epithelium persisting probably from the fetal period of life or developed from putative stem cells are a pathological condition which is not observed in healthy adult ovaries, and might be related to serous papillary adenocarcinoma manifestation in the adult ovarian surface epithelium. This observation needs attention to be further investigated.