Successful Delivery in a 39-Year-Old Patient with Anal Cancer after Fertility-Preserving Surgery Followed by Primary Chemoradiation and Low Anti-Mullerian Hormone Level.

Anal cancer was diagnosed in a 36-year-old nulliparous woman. Before chemoradiation was started, ovarian transposition and uterine fixation were performed in order to preserve capability for future parenthood. Despite of a low anti-Mullerian hormone level, IVF therapy was successful and resulted in a full-term delivery of a healthy baby by cesarean section. This is the first case report of successful pregnancy after full-dose pelvic radiation for anal cancer.

Initiation of ovarian stimulation independent of the menstrual cycle: a case-control study.

PURPOSE: In the GnRH-antagonist protocol, ovarian stimulation with gonadotropins typically commences on cycle day 2 or 3. Initiation of ovarian stimulation with a spontaneously occurring menstruation, however, poses significant organizational challenges for treatment centres and patients alike. It has previously been demonstrated in the context of fertility preservation that initiation of stimulation in the luteal phase is feasible in terms of retrieval of mature oocytes for cryopreservation. Herein, we report the extension of this concept to a routine IVF setting with the aim of establishing an ovarian stimulation protocol, which can be utilized independent of menstruation. Because of asynchrony of endometrium and embryo in such a setting, all fertilized oocytes have to be cryopreserved for a later transfer. METHODS: This was a prospective, case-control study (trial registration: NCT00795041) on the feasibility of starting ovarian stimulation in a GnRH-antagonist protocol in the luteal phase. Inclusion criteria were: IVF or ICSI; 18-36 years; ≤3 previous IVF/ICSI attempts; BMI 20-30 kg/m(2); regular cycle (28-35 days); luteal phase progesterone >7 ng/ml at initiation of stimulation. Exclusion criteria were: PCOS, endometriosis ≥AFS III°, unilateral ovary, expected poor response. Stimulation was performed with highly purified uFSH (Bravelle®) 300 IU/day and 0.25 mg/day GnRH-antagonist starting on cycle day 19-21 of a spontaneous menstrual cycle and commencing until hCG administration when three follicles ≥17 mm were present. All 2PN stage oocytes were vitrified for later transfers in programmed cycles. Feasibility was defined as the achievement of ongoing pregnancies progressing beyond the 12th gestational week in at least 2/10 study subjects (primary outcome). Secondary outcomes were gonadotropin consumption per oocyte obtained, stimulation duration, and fertilization rates. Study subjects were matched in a 1:3 ratio with concomitantly treated control cases of similar age, BMI, and duration of infertility who were treated in a conventional GnRH-antagonist protocol with 150-225 rFSH or HP-HMG/day. RESULTS: The study group consisted of ten subjects, mean age 31.4 years, BMI 25.4 kg/m(2), of which one had fertilization failure. Mean stimulation duration was 11.7 (SD 1.6) vs. 9.1 (SD 1.3) days, mean cumulative FSH dose was 3,495.0 (SD 447.5) vs. 2,040.5 (SD 576.2) IU, and mean number of oocytes was 8.8 (SD 5.0) vs. 10.0 (SD 5.4) in study vs. control group, respectively. Per follicle ≥10 mm, the cumulative FSH dose was 274.5 (SD 130.8) IU vs. 245.2 (SD 232.3) IU in study and control groups, respectively. Cumulative ongoing pregnancy rates were 1/10 (10 %) and 6/30 (20.0 %) in study and control group, respectively (difference: 10 %, 95 % confidence interval of the difference: -29.2-22.2 %, p = 0.47). Fertilization rate was similar between groups, with 63.5 % (SD 32.9) in the study and 61.3 % (SD 26.7) in the control group, respectively. Serum estradiol levels were significantly lower on the day of triggering final oocyte maturation with 1,005.3 (SD 336.2) vs. 1,977.4 pg/ml (SD 1,106.5) in study and control group, respectively. Similarly, peak estradiol biosynthesis per growing follicle ≥10 mm was lower in the study group (134 pg/ml, SD 158.4 vs. 186.7 pg/ml, SD 84.7). CONCLUSIONS: Per retrieved oocyte, a nearly threefold higher dose of FSH had to be administered when ovarian stimulation had been initiated in the luteal phase. Furthermore, the present study casts doubt on the efficacy of initiating ovarian stimulation in the luteal phase in terms of pregnancy achievement. Thus, this concept is currently not feasible for routine use, and it should also be explored further before using it at larger scale in the context of emergency stimulation for fertility preservation.

Serum 25-hydroxyvitamin D levels in patients with vulvar cancer.

BACKGROUND: The anticarcinogenic potential of vitamin D 25(OH)D has been attributed to the inhibition of proliferation of cells from different carcinomas. Reduced serum levels of 25(OH)D are associated with an increased incidence of various types of cancer. The influence of serum 25(OH)D on the incidence and outcome of patients with vulvar cancer is unknown. PATIENTS AND METHODS: The serum 25(OH)D levels in 24 patients with vulvar cancer and 24 age-matched cancer-free patients was investigated. The blood samples were collected between October 2009 and September 2010 and time of blood collection of each patient and control was matched to avoid seasonal variations between the pairs. RESULTS: The median 25(OH)D serum levels in the under 50 year old group of patients were significantly lower in the vulvar cancer group than the controls. The younger cancer group also had an age-related trend of lower median serum level than the older population. In the control population the trend was vice versa, yet this finding was not statistically significant. CONCLUSION: Serum 25(OH)D has a possible role in the pathogenesis and progression of vulvar cancer, but further investigations of the association of vitamin D and vulvar cancer as well as regarding its influence on patient survival and quality of life are warranted in the future.

Vitamin D receptor expression in patients with vulvar cancer.

The anticarcinogenic potential of vitamin D is attributed to antiproliferative and prodifferentiative effects on cells for a wide variety of carcinomas. The biological effects of 1,25(OH)(2)D (calcitriol) are mediated through a soluble receptor protein termed vitamin D receptor (VDR). However, thus far there have been no studies evaluating the association between VDR expression and vulvar cancer. Using immunohistochemical analysis, VDR expression was evaluated separately in the nucleus, cytoplasm and membrane, in vulvar cancer samples and adjacent non-pathological vulvar tissue from 48 squamous cell carcinoma patients with no prior therapy, and the association between VDR and overall survival was investigated. Overall, among the 48 vulvar cancer cases, nuclear and cytoplasmic VDR expression was present in 47 (97.9%) and 23 (47.9%) cases respectively. The median nuclear VDR expression was significantly higher as compared to the cytoplasmic VDR in the vulvar cancer tissue. No significant correlation between VDR values and the age of the patients was detected. Nuclear and cytoplasmatic VDR in the vulvar cancer tissue were also compared according to the tumor size, and no significant association between mean tumor VDR and tumor size was detected. There was no association between cytoplasmatic VDR expression and OS, but better OS was observed in patients with reduced nuclear VDR expression as compared to those with high VDR expression. VDR may be considered as a useful pathological marker.

Influence of calcitriol on prostaglandin- and vitamin D-metabolising enzymes in benign and malignant breast cell lines.

BACKGROUND: Cyclooxygenase-2 (COX-2) is a potential molecular prognostic factor for breast cancer, and calcitriol [1,25(OH)(2)D(3)], the biologically active form of vitamin D, is a promising target in breast cancer therapy. MATERIALS AND METHODS: The influence of calcitriol on the proliferation and the effects of calcitriol on the expression of prostaglandin- and vitamin D-metabolising enzymes were examined in benign and malignant breast cells. RESULTS: Calcitriol inhibited the proliferation of MCF-10F and MCF-7 cells but not of invasive MDA-MB-231 cells and reduced the expression of COX-2 and 15-hydroxyprostaglandin dehydrogenase (15-PGDH) in the benign breast cell line MCF-10F. Furthermore, dysregulation in vitamin D-metabolising proteins was detected, especially in MDA-MB-231 cells. CONCLUSION: These results suggest dysregulation of vitamin D metabolism and a lack of a possible influence of calcitriol on the metabolism of prostaglandins in the malignant breast cell lines.

Expression of prostaglandin- and vitamin D-metabolising enzymes in benign and malignant breast cells.

BACKGROUND: Cyclooxygenase-2 (COX-2) plays a crucial role in prognosis of malignancy and has been associated with carcinogenesis, particularly neoangiogenesis and tumor progression. 15-Hydroxyprostaglandin dehydrogenase (15-PGDH) is described as a tumour suppressor in cancer. The antiproliferative effects of calcitriol [1,25(OH)(2)D(3)] mediated via the vitamin D receptor (VDR) render vitamin D a promising target in breast cancer therapy. MATERIALS AND METHODS: The expression of prostaglandin (PG)-metabolizing enzymes, vitamin D-metabolising enzymes and VDR were determined in benign and malignant breast cell lines using western blot analysis. RESULTS: We detected an inverse correlation between the two types of metabolism, a reduced VDR expression in the malignant breast cell lines, and therefore an insufficient induction of 24-hydroxylase in the malignant cells. CONCLUSION: We suggest the possibility of dysregulation of vitamin D-metabolizing enzymes in malignant breast cell lines.

Vitamin D, calcidiol and calcitriol regulate vitamin D metabolizing enzymes in cervical and ovarian cancer cells.

BACKGROUND: The vitamin D metabolizing enzymes 25-, 1α- and 24-hydroxylase are expressed in malignant cells of the cervix and the ovaries. The aim of this study was to obtain further information about the regulation of the aforementioned enzymes by vitamin D, calcidiol and calcitriol in cervical and ovarian cancer. MATERIALS AND METHODS: The human cervical adenocarcinoma cell line HeLa and the human ovarian adenocarcinoma cell line OVCAR-3 were incubated with vitamin D, calcidiol and calcitriol. The influence of vitamin D and its metabolites on the expression of 25-, 1α- and 24-hydroxylase was assessed by real-time RT-PCR. RESULTS: Calcitriol significantly increased the 24-hydroxylase mRNA levels in HeLa and OVCAR-3 cells. The expression of 25- and 1α-hydroxylase was not regulated in a statistically significant manner. CONCLUSION: These results suggest that in HeLa as well as OVCAR-3 cell lines, the metabolism of vitamin D is regulated via the expression of the catabolizing 24-hydroxylase.

[The diagnostic of male infertility - an important part of reproductive medicine]. / Die Diagnostik der männlichen Infertilität - ein wichtiger Bestandteil der Reproduktionsmedizin.

Male infertility is frequently involved in the couples infertility and therefore the diagnostic work up of the couple should always involve gynaecologists and andrologists. The main task of the interdisciplinary diagnostic work up is the direction of the couple to potential treatments, bearing in mind that spontaneous pregnancies occur frequently in infertile couples. Well established pathways for the male diagnostic work up in infertility exist only marginally. A minimal andrological evaluation should be performed at least in all infertile men after one year of unsuccessful unprotected intercourse or earlier if established male or female risk factors are present. Components of the minimal evaluation of the male partner couple should include at least a reproductive history and two semen analyses according to WHO standards. An evaluation by an andrologist should be done as routine procedure. However, a full andrological evaluation is especially important if the initial evaluation demonstrates an abnormal male reproductive history or an abnormal semen analysis. Further evaluation of the male partner should also be considered in couples with unexplained infertility and in couples in whom there is a treated female factor and persistent infertility. In addition to the requirements of a minimal evaluation, a full evaluation for male infertility should include in addition at least a physical and genital examination. Based on the results of the andrological evaluation, the physician may recommend additional evaluations. These additional evaluations may include an endocrine evaluation, ultrasonography of the scrotal content and/or prostate and seminal vesicles, genetic screening and the conductance of a diagnostic/therapeutic testicular biopsy. Finally the diagnostic work up of the male infertile patient will lead to a solid dia-gnosis on which the subsequent therapeutic procedures must be based.

Expression of prostaglandin metabolising enzymes COX-2 and 15-PGDH and VDR in human granulosa cells.

BACKGROUND: Prostaglandins (PGs) within the periovulatory follicle are essential for various female reproductive functions such as follicular development and maturation. In animal models, granulosa cells express the PG synthesizing enzyme cyclooxygenase-2 (COX-2) and the PG inactivating enzyme 15-hydroxyprostaglandin dehydrogenase (15-PGDH). First references suggest a correlation between vitamin D and prostaglandin metabolism through the impact of 1,25(OH)2D3 (calcitriol) on the expression of COX-2 and 15-PGDH. MATERIALS AND METHODS: The expression of COX-2, 15-PGDH and the vitamin D receptor (VDR) in human granulosa cells (COV434, hGC and HGL5), which were originally isolated from different stages of follicular maturation, was determined by real-time PCR (RT-PCR) and Western blot analysis. RESULTS: A positive correlation of COX-2 and VDR protein was found in the COV434 and HGL5 cells and an inverse correlation of 15-PGDH and VDR protein levels in all the investigated cell types. CONCLUSION: There may be a link between VDR, associated target genes and prostaglandin metabolism in human follicular maturation and luteolysis.

Prostaglandin metabolizing enzymes in correlation with vitamin D receptor in benign and malignant breast cell lines.

BACKGROUND: The antiproliferative effects of calcitriol (1,25(OH)2D3) mediated via the vitamin D receptor (VDR), render the biologically active form of vitamin D a promising target in breast cancer therapy. Furthermore, breast cancer is associated with inflammatory processes based on an up-regulation of cyclooxygenase-2 (COX-2) expression, the prostaglandin E2 (PGE2) synthesizing enzyme. The PGE2 metabolizing enzyme, 15-hydroxyprostaglandin dehydrogenase (15-PGDH) is described as a tumor suppressor in cancer. First references suggest a correlation between vitamin D and prostaglandin metabolism through the impact of 1,25(OH)2D3 on the expression of COX-2 and 15-PGDH. MATERIALS AND METHODS: The expression of VDR, COX-2 and 15-PGDH in benign MCF-10F and malignant MCF-7 breast cells was determined by real-time PCR (RT-PCR) and Western blot analysis. RESULTS: Although the RT-PCR data were divergent from those obtained from the Western blot analysis, the COX-2 protein expression was MCF-7 2-fold higher in the MCF-7 compared to the MCF-10F cells. Moreover, a correlation of 15-PGDH to VDR by RT-PCR was found in both cell lines. The VDR protein levels were inversely correlated to the 15-PGDH protein levels and revealed that the MCF-10F cells had the highest VDR expression. CONCLUSION: A possible link between VDR-associated target genes and prostaglandin metabolism is suggested.

25-Hydroxyvitamin D3 1alpha-hydroxylase splice variants in benign and malignant ovarian cell lines and tissue.

Calcitriol is judged to have a positive effect on control of the immune system, cell growth and differentiation and therefore, the prevention of cancer genesis. The aim of this study was to detect any possible differences in the 25-hydroxyvitamin D-1alpha-hydroxylase (1alphaOHase)-expression between benign and malignant ovarian tissue and cell lines. The analysis was conducted quantitatively, by means of nested "touchdown" PCR and Western blot, and qualitatively, with the use of real-time PCR and Western blot. The gene structure was sequenced. Compared to the benign cell line, the malignant cell lines showed a significantly higher expression of 1alphaOHase at the RNA level. A statistically lower expression of the 1alphaOHase protein was found in the malignant tissue. In the malignant cell lines and tissues, divergent bands were detected, which led to various splice variants on sequencing. Their increased expression in malignancy is possibly bound to the reduction of enzyme activity, which may lead to the genesis of ovarian cancer. In the future, preventive and therapeutic activities may result from these findings.

Expression of 25-hydroxyvitamin D3-24-hydroxylase in benign and malignant ovarian cell lines and tissue.

Calcitriol (1,25-dihydroxyvitamin D3, 1,25(OH)2D3) plays a pivotal role in maintaining calcium and phosphate homeostasis. Aside from that, it supports the native and attenuates the acquired immune system and has positive effects on cell growth, differentiation and the prevention of carcinogenesis. The goal of this study was to detect possible differences in the expression of the calcitriol-degrading enzyme 24-hydroxylase (24-OHase) between malignant and benign ovarian cell lines and tissue. The analyses were based on real-time PCR, nested touchdown PCR and Western blot. When compared to benign granulose GLZ cells, the malignant HGL5 cells showed a significantly higher 24-OHase expression at the protein level (p<0.01). In the malignant ovarian tissue, the expression was significantly higher in RNA (p<0.001), but lower at the protein level (p<0.01). An increased 24-OHase expression could indicate a decrease in available calcitriol.

Analysis of ventricular hypertrabeculation and noncompaction using genetically engineered mouse models.

Ventricular trabeculation and compaction are two of the many essential steps for generating a functionally competent ventricular wall. A significant reduction in trabeculation is usually associated with ventricular compact zone deficiencies (hypoplastic wall), which commonly lead to embryonic heart failure and early embryonic lethality. In contrast, hypertrabeculation and lack of ventricular wall compaction (noncompaction) are closely related defects in cardiac embryogenesis associated with left ventricular noncompaction, a genetically heterogeneous disorder. Here we summarize our recent findings through the analyses of several genetically engineered mouse models that have defects in cardiac trabeculation and compaction. Our data indicate that cellular growth and differentiation signaling pathways are keys in these ventricular morphogenetic events.

Expression of 25 hydroxyvitamin D3-1alpha-hydroxylase in human endometrial tissue.

1,25(OH)(2)D(3) (calcitriol) has been shown to play an important role in cell proliferation, differentiation and immune responsiveness. The enzyme responsible for calcitriol synthesis 25 hydroxyvitamin D(3)-1alpha-hydroxylase (1alpha-OHase) has been reported in many human tissues. The aim of this study was to investigate the expression of 1alpha-OHase in gynaecological tissues. Using a highly specific nested touchdown PCR we examined the expression of 1alpha-OHase in normal and malignant endometrial tissue and in human endometrial Ishikawa cells. In addition, we analyzed the protein expression of 1alpha-OHase by Western blot. The expression of 1alpha-OHase in normal and malignant endometrial tissue and Ishikawa cells was detected and splice variants of the enzyme in Ishikawa cells were identified. These data suggest an alternative splicing of 1alpha-OHase in malignant endometrial tissue and cells. We postulate that the expression of 1alpha-OHase gene variants may contribute to the antiproliferative effects of calcitriol. In conclusion, the modulation of the 1alpha-OHase opens up a new target for vitamin D(3) related therapies in endometrial cancer.

Analysis of 25-hydroxyvitamin D3-1alpha-hydroxylase in normal and malignant breast tissue.

BACKGROUND: The presence of extra-renal 25-hydroxyvitamin D3 [25(OH)D3]-1alpha-hydroxylase (1alpha-OHase) has been reported in several cell types including prostate and colon cancer cells. Additionally, alterations in the local production of 1alpha,25-dihydroxyvitamin D [1alpha,25(OH)2D3] have been implicated in the tumorigenesis of these malignancies. The aim of this study was to analyze whether normal breast tissue or breast cancer cells expressed 1alpha-OHase and to evaluate whether breast tissue possessed the capacity to produce 1alpha,25(OH)2D3 from 25(OH)D3. MATERIALS AND METHODS: Total RNA was extracted from normal breast tissue (n = 11), breast carcinomas (n = 12) and cultured MCF-7 breast cancer cells for real-time (LightCycler using specific hybridization probes) and conventional PCR analysis. RESULTS: mRNA for 1alpha-OHase was detected in breast cancer tissue and in MCF-7 breast cancer cells. Interestingly, the mRNA levels for 1alpha-OHase were significantly increased in breast cancer compared to normal breast tissue. When the MCF-7 cells were treated with 1alpha,25(OH)2D3, cell proliferation was inhibited in a dose-dependent manner. Incubation of the MCF-7 cells with [3H]-25(OH)D3 resulted in its conversion to [3H]-1,25(OH)2D3. The 1alpha-OHase activity in the MCF-7 cells was blocked by a specific cytochrome P450 inhibitor, clotrimazole. CONCLUSION: The data suggest that at least breast cancer cells expressed 1alpha-OHase mRNA and, therefore, might have the ability to synthesize 1alpha,25(OH)2D3 within the cells. The local production of 1alpha,25(OH)2D3 might play an important role in regulating the proliferation and differentiation of breast cells. We hypothesize that alterations in the local production of 1alpha,25(OH)2D3 may be involved in the tumorigenesis of breast cancer. Additionally, breast cancer may be a target for treatment with precursors of biologically-active vitamin D analogs.

Vitamin D--metabolism in the human breast cancer cell line MCF-7.

BACKGROUND: The three main vitamin D metabolizing enzymes, vitamin D3-25-hydroxylase (25-OHase, 25-hydroxylase), 25-hydroxyvitamin D3-1alpha-hydroxylase (1alpha-OHase, 1alpha-hydroxylase) and 25-hydroxyvitamin D3-24-hydroxylase (24-OHase, 24-hydroxylase), have been described in malignant breast tissue. This in vitro study aimed to obtain more information regarding the regulation of these enzymes in the human breast cancer cell line MCF-7. MATERIALS AND METHODS: Vitamin D receptor (VDR)- positive MCF-7 cells in culture were stimulated with the vitamin D metabolites vitamin D3, 25-hydroxyvitamin D3 and 1,25-dihydroxyvitamin D3 for 24, 48; 72 and 96 hours in physiological and supraphysiological concentrations. The expressions of 25-hydroxylase, 1alpha-hydroxylase and 24-hydroxylase and their changes after stimulation were assessed by real-time PCR. RESULTS: The expression of 25-hydroxylase was slightly influenced by vitamin D3. The expression of 1alpha-hydroxylase was induced after stimulation with vitamin D3 and 25-hydroxyvitamin D3. Stimulation with 1,25-dihydroxyvitamin D3 markedly increased the expression of 24-hydroxylase time- and dose-dependently. CONCLUSION: Our results demonstrated that MCF-7 cells are able to regulate the expression of 24-hydroxylase. This might be a mechanism for these tumor cells to protect themselves against the antiproliferative and apoptosis-inducing effects of calcitriol.