Revascularization of human ovarian grafts is equally efficient from both sides of the cortex tissue.

RESEARCH QUESTION: Does revascularization of human ovarian grafts in a mouse model occur with equal efficiency from both sides of the cortex tissue? DESIGN: Twenty-four frozen-thawed ovarian cortex pieces from 12 women were transplanted to immunodeficient mice, for 8 days to analyse graft revascularization using immunohistochemical detection of murine CD31, or for 8 weeks to evaluate follicle density (follicles/mm3). The CD31-positive vessel area and density were quantified using a custom-designed application. Three regions of interest (ROI) were defined in each tissue section: the cortical side, the centre and the medullary side. Vessels were subdivided into three categories according to size: microvessels (<300 µm2), small vessels (300-1000 µm2) and large vessels (>1000-3000 µm2). RESULTS: No significant difference in the mean percentage of the CD31-positive vessel area was found between the three ROI (cortical side: 3.9% ± 0.2%; centre: 3.5% ± 0.2%; medullary side: 4.0% ± 0.3%; P = 0.17), but a significantly lower density of vessels was found in the centre of the human ovarian grafts compared with the cortical and medullary sides (cortical side: 323 ± 14 vessels/mm2; centre: 240 ± 12 vessels/mm2; medullary side: 301 ± 18 vessels/mm2; P < 0.001). Microvessels comprised 89-91% of all vessels in the three ROI. Follicle density in ungrafted cortex pieces was 51.8 ± 17.3 and 14.7 ± 3.7 follicles/mm3 after 8 weeks of xenografting, resulting in a follicle survival rate of 28%. CONCLUSIONS: Host revascularization was established equally efficiently from both sides of transplanted human ovarian cortex, suggesting that transplantation techniques ensuring revascularization from both sides of the ovarian graft could potentially facilitate faster graft revascularization.

The precise ovarian volume is significantly associated with serum concentrations of antimüllerian hormone, the luteinizing hormone/follicle-stimulating hormone ratio, and total testosterone.

OBJECTIVE: To evaluate potential associations between concentrations of antimüllerian hormone (AMH) and T as well as the LH/FSH ratio and the unbiased precise ovarian volume obtained after unilateral ovariectomy. DESIGN: Cohort study. SETTING: University hospital. PATIENT(S): A total of 765 patients having one ovary surgically removed for fertility preservation. Inclusion criteria were age >15 years and ovarian volume <25 mL; 386 women had one or more hormone parameter (AMH, LH, FSH, or total T) determined before oophorectomy. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): A precise weight of the ovary was equated with ovarian volume. Associations between ovarian volume and AMH, the LH/FSH ratio, T concentrations, and body mass index (BMI) were evaluated. Patient characteristics in relation to ovarian volume cutoff values between 8 and 12 mL were also examined. RESULT(S): Ovarian volume was significantly positively associated with concentrations of AMH, the LH/FSH ratio, and T. Ovarian volume, concentrations of AMH and LH, and the LH/FSH ratio were significantly augmented in women having ovarian volumes above a threshold of 8, 9, and 10 mL compared with those below. Average age, FSH, and T concentrations did not differ between below and above the 10 mL threshold. There was a significant association between BMI and ovarian volume and BMI and T, while other hormone parameters were nonsignificant. CONCLUSION(S): The precise ovarian volume reflected ovarian activity measured as circulating concentrations of AMH and T as well as the LH/FSH ratio. These significant associations showed continuous progression, and a 10 mL threshold offered no clear difference compared with other volume threshold values.

Reduced hepatic metallothionein expression in first trimester fetuses in response to intrauterine smoking exposure: a consequence of low maternal zinc levels?

STUDY QUESTION: Does maternal smoking in early pregnancy affect metallothionein 1 and 2 (MT1 and MT2) mRNA and protein expression in first trimester placenta or embryonic/fetal liver? SUMMARY ANSWER: In the first trimester, MT protein expression is seen only in liver, where smoking is associated with a significantly reduced expression. WHAT IS KNOWN ALREADY: Zinc homeostasis is altered by smoking. Smoking induces MT in the blood of smokers properly as a result of the cadmium binding capacities of MT. In term placenta MT is present and smoking induces gene and protein expression (MT2 in particular), but the MT presence and response to smoking have never been examined in first trimester placenta or embryonic/fetal tissues. STUDY DESIGN, SIZE, DURATION: Cross sectional study where the presence of MT mRNA and protein was examined at the time of the abortion. The material was collected with informed consent after surgical intervention and frozen immediately. For protein expression analysis, liver tissue originating from smoking exposed n = 10 and unexposed n = 12 pregnancies was used. For mRNA expression analyses, placental tissue originating from smokers n = 19 and non-smokers n = 23 and fetal liver tissue from smoking exposed n = 16 and smoking unexposed pregnancies n = 13, respectively, were used. PARTICIPANTS/MATERIALS, SETTING, METHODS: Tissues were obtained from women who voluntarily and legally chose to terminate their pregnancy between gestational week 6 and 12. Western blot was used to determine the protein expression of MT, and real-time PCR was used to quantify the mRNA expression of MT2A and eight MT1 genes alongside the expression of key placental zinc transporters: zinc transporter protein-1 (ZNT1), Zrt-, Irt-related protein-8 and -14 (ZIP8 and ZIP14). MAIN RESULTS AND THE ROLE OF CHANCE: A significant reduction in the protein expression of MT1/2 in liver tissue (P = 0.023) was found by western blot using antibodies detecting both MT forms. Overall, a similar tendency was observed on the mRNA level although not statistically significant. Protein expression was not present in placenta, but the mRNA regulation suggested a down regulation of MT as well. A suggested mechanism based on the known role of MT in zinc homeostasis could be that the findings reflect reduced levels of easily accessible zinc in the blood of pregnant smokers and hence a reduced MT response in smoking exposed fetal/embryonic tissues. LIMITATIONS AND REASONS FOR CAUTION: Smoking was based on self-reports; however, our previous studies have shown high consistency regarding cotinine residues and smoking status. Passive smoking could interfere but was found mainly among smokers. The number of fetuses was limited, and other factors such as medication and alcohol might affect the findings. Information on alcohol was not consistently obtained, and we cannot exclude that it was more readily obtained from non-users. In the study, alcohol consumption was reported by a limited number (less than 1 out of 5) of women but with more smokers consuming alcohol. However, the alcohol consumption reported was typically limited to one or few times low doses. The interaction between alcohol and smoking is discussed in the paper. Notably we would have liked to measure zinc status to test our hypothesis, but maternal blood samples were not available. WIDER IMPLICATIONS OF THE FINDINGS: Zinc deficiency-in particular severe zinc deficiency-can affect pregnancy outcome and growth. Our findings indicate that zinc homeostasis is also affected in early pregnancy of smokers, and we know from pilot studies that even among women who want to keep their babies, the zinc status is low. Our findings support that zinc supplements should be considered in particular to women who smoke. STUDY FUNDING/COMPETING INTEREST(S): We thank the Department of Biomedicine for providing laboratory facilities and laboratory technicians and the Lundbeck Foundation and Læge Sofus Carl Emil Friis og Hustru Olga Doris Friis Legat for financial support. The authors have no competing interests to declare. TRIAL REGISTRATION NUMBER: N/A.

Changes in first trimester fetal CYP1A1 and AHRR DNA methylation and mRNA expression in response to exposure to maternal cigarette smoking.

Prenatal exposure to maternal cigarette smoking increases the risk of intrauterine growth retardation, adverse pregnancy outcomes, and diseases later in life. Exposure can result in postnatal global and gene-specific DNA methylation changes, with the latter well documented for the CYP1A1 and AHRR genes involved in the detoxification of xenobiotic substances. This study assessed the impact of exposure to maternal smoking on first trimester fetal CYP1A1 and AHRR mRNA expression and DNA methylation for CpG-sites displaying maternal smoking during pregnancy-mediated methylation changes at birth. The analyses included first trimester (6-12 weeks) placentas (N=39) and livers (N=43). For AHRR, exposure to maternal smoking was associated with increased DNA methylation in the placentas of female fetuses; mRNA expression, however, was unchanged. While exposure to maternal smoking was not associated with AHRR DNA methylation changes in fetal livers; mRNA expression was increased. For CYP1A1, exposure to maternal smoking was not associated with fetal DNA methylation changes whereas mRNA expression increased in placentas and male fetal livers. These results show that first trimester exposure to maternal smoking is associated with CYP1A1 and AHRR DNA methylation and mRNA expression changes. However, the results also indicate that maternal smoking during pregnancy-mediated postnatal CYP1A1 and AHRR DNA methylation changes are not imprinted during the first trimester.

Temporal expression pattern of genes during the period of sex differentiation in human embryonic gonads.

The precise timing and sequence of changes in expression of key genes and proteins during human sex-differentiation and onset of steroidogenesis was evaluated by whole-genome expression in 67 first trimester human embryonic and fetal ovaries and testis and confirmed by qPCR and immunohistochemistry (IHC). SRY/SOX9 expression initiated in testis around day 40 pc, followed by initiation of AMH and steroidogenic genes required for androgen production at day 53 pc. In ovaries, gene expression of RSPO1, LIN28, FOXL2, WNT2B, and ETV5, were significantly higher than in testis, whereas GLI1 was significantly higher in testis than ovaries. Gene expression was confirmed by IHC for GAGE, SOX9, AMH, CYP17A1, LIN28, WNT2B, ETV5 and GLI1. Gene expression was not associated with the maternal smoking habits. Collectively, a precise temporal determination of changes in expression of key genes involved in human sex-differentiation is defined, with identification of new genes of potential importance.

Hallmarks of Human Small Antral Follicle Development: Implications for Regulation of Ovarian Steroidogenesis and Selection of the Dominant Follicle.

Regulation of human ovarian steroidogenesis differs from other species and precise knowledge on how human small antral follicles (hSAF) develop and acquire competence for continued growth and steroid output is still incomplete. The present study has characterized almost 1,000 normal hSAF collected in connection with cryopreservation of ovarian tissue for fertility preservation. The antral follicles (ranging from 3 to 13 mm) were generally aspirated from one ovary surgically removed during the natural cycle, and the follicular fluid (FF) and the granulosa cells (GC) were isolated and snap-frozen. In FF, the following hormones were measured: inhibin-B, inhibin-A, AMH, follistatin, PAPP-A, estradiol, progesterone, testosterone, and androstenedione. In GC, mRNA gene expressions using q-PCR were measured for the following genes: FSHR, AMH, CYP19, and AR. All samples in which one of the abovementioned parameters was measured were included, but typically multiple parameters were measured. Highly significant differences in concentration and follicular content in relation to follicular diameter were found for all measured hormones despite massive variability in-between follicles for any given diameter. The results demonstrate that profound changes take place in the hormonal microenvironment around follicular diameters of 8-11 mm corresponding to when follicular selection occurs. At this point, inhibin-B and inhibin-A showed distinct peaks concomitant with a significant reduction in both AMH protein and mRNA expression. Concentrations of inhibins, androgens, FSHR, and AR were intimately associated, and it is suggested that inhibin-B in combination with PAPP-A and thereby IGF2 activity exerts important paracrine signaling at follicular selection. At the same time upregulation of estradiol synthesis and CYP19 mRNA expression increased steroid output profoundly. Furthermore, the highly significant association between FSHR and AR mRNA gene expression enforces important functions of androgens in follicular development. Collectively, these data reintroduce the understanding of the follicular phase as two parted in which regulation of steroidogenesis differs. The profound changes taking place around follicular selection highlight important paracrine actions of TGF-ß family members and IGFs for securing dominance of the selected follicle.

Assessment of global DNA methylation in the first trimester fetal tissues exposed to maternal cigarette smoking.

AIMS: Maternal cigarette smoking during pregnancy increases the risk of negative health consequences for the exposed child. Epigenetic mechanisms constitute a likely link between the prenatal exposure to maternal cigarette smoking and the increased risk in later life for diverse pathologies. Maternal smoking induces gene-specific DNA methylation alterations as well as global DNA hypermethylation in the term placentas and hypomethylation in the cord blood. Early pregnancy represents a developmental time where the fetal epigenome is remodeled and accordingly can be expected to be highly prone to exposures with an epigenetic impact. We have assessed the influence of maternal cigarette smoking during the first trimester for fetal global DNA methylation. METHODS AND RESULTS: We analyzed the human fetal intestines and livers as well as the placentas from the first trimester pregnancies. Global DNA methylation levels were quantified with ELISA using a methylcytosine antibody as well as with the bisulfite pyrosequencing of surrogate markers for global methylation status, LINE-1, and AluYb8. We identified gender-specific differences in global DNA methylation levels, but no significant DNA methylation changes in exposure responses to the first trimester maternal cigarette smoking. CONCLUSIONS: Acknowledging that only examining subsets of global DNA methylation markers and fetal sample availability represents possible limitations for the analyses, our presented results indicate that the first trimester maternal cigarette smoking is not manifested in immediate aberrations of fetal global DNA methylation.

Effect of first line cancer treatment on the ovarian reserve and follicular density in girls under the age of 18 years.

OBJECTIVE: To study the impact of first-line antineoplastic treatment on ovarian reserve in young girls returning for ovarian tissue cryopreservation (OTC) in connection with a relapse. DESIGN: Retrospective case-control study. SETTING: University hospitals. PATIENT(S): Sixty-three girls under the age of 18 years who underwent OTC before (group 1: 31 patients) and after (group 2: 32 patients) their initial cancer treatment. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Follicular densities (follicles/mm3) measured from an ovarian cortical biopsy before OTC. The ovarian volume (mL) of entire ovaries excised for OTC was also monitored. RESULT(S): There was no statistically significant difference in the mean age or follicular density between groups 1 and 2 (334 ± 476/mm3 vs. 327 ± 756/mm3). In contrast, the ovarian volume and total number of ovarian cortex chips cryopreserved were statistically significantly lower in patients who received gonadotoxic treatment before OTC (mean ± standard deviation [SD]: ovarian volume, 5.3 ± 3.1 mL vs. 2.9 ± 2.1 mL, respectively; number of cortex chips: 21.3 ± 8.1 vs. 15.2 ± 7.1, respectively). The reduction in the estimated ovarian reserve ranged from 10% to 20% in children to around 30% in adolescent girls (>10 years). CONCLUSION(S): Girls under the age of 10 tolerate a gonadotoxic insult better than adolescents, who may experience up to a 30% reduction in the ovarian reserve via first-line gonadotoxic treatment, which at present is considered to have little effect on the follicle pool. This information will improve counseling of young female cancer patients in deciding whether to undergo fertility preservation treatment.