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

The Child Cancer Foundation in Denmark, The Novo Nordisk Foundation and the EU interregional project ReproHigh/ReproUnion are thanked for having funded this study.Objective: To study the impact of first-line antineoplastic treatment on the ovarian reserve in young girls returning for ovarian tissue cryopreservation (OTC) in connection with a relapse. Design: Retrospective case-control study. Setting: University hospitals. Patients: 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.PostprintPeer reviewe

Expression and Role of INSL3 in the Fetal Testis

Insulin-like peptide 3 (INSL3) is a small peptide hormone of the insulin-relaxin family which is produced and secreted by the fetal Leydig cells in the testes only. It appears to be undetectable in female fetuses. In the human fetus INSL3 synthesis begins immediately following gonadal sex determination at weeks 7 to 8 post coitum and the peptide can be detected in amniotic fluid 1 to 2 weeks later. INSL3 acts through a unique G-protein-coupled receptor, called RelaXin-like Family Peptide receptor 2 (RXFP2), which is expressed by the mesenchymal cells of the gubernacular ligament linking the testes to the inguinal wall. The role of INSL3 in the male fetus is to cause a thickening of the gubernaculum which then retains the testes in the inguinal region, while the remainder of the abdominal organs grow away in an antero-dorsal direction. This represents the first phase of testis descent and is followed later in pregnancy by the second inguino-scrotal phase whereby the testes pass into the scrotum through the inguinal canal. INSL3 acts as a significant biomarker for Leydig cell differentiation in the fetus and may be reduced by maternal exposure to endocrine disrupting chemicals, such as xenoestrogens or phthalates, leading to cryptorchidism. INSL3 may have other roles within the fetus, but as a Leydig cell biomarker its reduction acts also as a surrogate for anti-androgen action

Expression of the Insulin-like Growth Factor system in first and second trimester human embryonic and fetal gonads

Financial Support: This work was supported by The Medical Research Council [MR/L010011/1 to PAF] and the European Community's Seventh Framework Programme (FP7/2007-2013) [under grant agreement no 212885 to PAF], BBSRC/EASTBIO (to AZ), ESHRE supported the ReproUnion fellowship (to AZ), Rigshospitalets Forskningspuljer (to LSM), and ReproUnion 1.0 (to LSM). Acknowledgement Marianne Sguazzino is acknowledged for excellent technical assistance. Gabriela Gudbergsen is acknowledged for her excellent design of Fig. 1. Data Availability: The dataset generated and/or analyzed during the current study are not publicly available but are available from the corresponding author on reasonable request. Microarray data are available with the Array Express accession number: E-MTAB-5611Peer reviewedPostprin

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

Abstract 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

Cryopreservation of ovarian tissue may be considered in young girls with galactosemia

This study was founded by Rigshospitalets Forskingspuljer, the Novo Nordisk Foundation, and ReproUnion.Purpose. The aim was to describe the first experience with fertility preservation by cryopreservation of ovarian tissue (OTC) in pre-pubertal girls with galactosemia and further to characterize ovarian follicular morphology and expression of proteins important for ovarian function. Methods. Retrospectively, follicle density was estimated in ovarian cortical tissues from 6 pre-pubertal girls below the age of 12 years diagnosed with galactosemia and from 31 girls below the age of 18 years who had one ovary removed for fertility preservation for other reasons prior to gonadotoxic treatment. Additionally, expression of 4 glycoproteins important for follicle development were analyzed with immunohistochemistry in two galactosemic ovaries (aged 0.9 and 1.7 years) and compared to normal age-matched controls. The proteins included were: anti-Müllerian hormone (AMH) pro-mature and C-terminal, growth differentiation factor-9 (GDF-9), bone morphogenetic protein 15 (BMP-15), and pregnancy-associated plasma protein A (PAPP-A). Results. Girls with galactosemia below the age of 5 years presented with morphological normal follicles and follicle densities within the 95% confidence interval (CI) of controls. No follicles were detected in the ovary from an 11.7-year-old girl with galactosemia. Expression of AMH, GDF-9, BMP-15, and PAPP-A appeared similar in follicles from girls with galactosemia and controls. Conclusions. These findings suggest that young girls with galactosemia maintain follicles in early childhood and fertility cryopreservation may be considered an option in this patient group. The pathophysiology of galactosemia leading to an accelerated follicle loss is unknown and it is currently unknown to what extent transplanted ovarian tissue can sustain fertility in adult life.PostprintPeer reviewe

Development and validation of a liquid chromatography-tandem mass spectrometry method for the determination of nicotine and its metabolites in placenta and umbilical cord.

Tobacco exposure during pregnancy is associated with obstetric and fetal complications. This study developed and validated a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to analyse the placenta (PL) and umbilical cord (UC) for nicotine, cotinine and hydroxycotinine (OH-cotinine). Specimens were homogenized in water, followed by solid-phase extraction. Chromatographic separation was performed using an Atlantis HILIC Silica column. Detection was accomplished in electrospray in positive mode. Method validation included: linearity (5 to 1000 ng/g), accuracy (86.9 to 105.2% of target concentration in PL, and 89.1 to 105.0% in UC), imprecision (6.8 to 11.8% in PL, and 7.6 to 12.2% in UC), limits of detection (2 ng/g for cotinine and OH-cotinine, and 5 ng/g for nicotine) and quantification (5 ng/g), selectivity (no endogenous or exogenous interferences), matrix effect (-34.1 to -84.5% in PL, %CV = 9.1-24.0%; -18.9 to -84.7% in UC, %CV = 10.2-23.9%), extraction efficiency (60.7 to 131.5% in PL, and 64.1 to 134.2% in UC), and stability 72 h in the autosampler ( < 11.5% loss in PL, and < 13% loss in UC). The method was applied to 14 PL and UC specimens from tobacco users during pregnancy. Cotinine (6.8-312.2 ng/g in PL; 6.7-342.3 ng/g in UC) was the predominant analyte, followed by OH-cotinine ( < LOQ-80.2 ng/g in PL; < LOQ-80.5 ng/g in UC) and nicotine (5.7-63.7 ng/g in PL; 5.1-63.3 ng/g in UC). In a future study, this method will be applied to more than 150 specimens collected from a wide clinical study to evaluate the usefulness of maternal hair, meconium, placenta and umbilical cord compared to the maternal interview to detect in utero drug exposure

Concentration of perfluorinated compounds and cotinine in human foetal organs, placenta, and maternal plasma

We thank The Research Pools of Rigshospitalet and the EU Interregional Project ReproUnion for funding this study.Background: Perfluoroalkyl substances (PFASs) are bio-accumulative pollutants, and prenatal exposure to PFASs is believed to impact human foetal development and may have long-term adverse health effects later in life. Additionally, maternal cigarette smoking may be associated with PFAS levels. Foetal exposure has previously been estimated from umbilical cord plasma, but the actual concentration in foetal organs has never been measured. Objectives: The concentrations of 5 PFASs and cotinine – the primary metabolite of nicotine – were measured in human foetuses, placentas, and maternal plasma to evaluate to what extent these compounds were transferred from mother to foetus and to determine if the PFAS concentrations were associated with maternal cigarette smoking. Methods: Thirty-nine Danish women who underwent legal termination of pregnancy before gestational week 12 were included; 24 maternal blood samples were obtained together with 34 placental samples and 108 foetal organs. PFASs and cotinine were assayed by liquid chromatography/triple quadrupole mass spectrometry. Results:  In foetal organs, the average concentrations of perfluorooctanesulphonic acid (PFOS), perfluorooctanoic acid (PFOA), perfluorononanoic acid (PFNA), perfluoroundecanoic acid (PFUnDa), and perfluorodecanoic acid (PFDA) were 0.6 ng/g, 0.2 ng/g, 0.1 ng/g, 0.1 ng/g, and 0.1 ng/g, respectively. A significant positive correlation was found between the exposure duration, defined as foetal age, and foetal to maternal ratio for all five PFASs and cotinine. Smokers presented 99 ng/g cotinine in plasma, 108 ng/g in placenta, and 61 ng/g in foetal organs. No correlation between the maternal cotinine concentrations and PFAS concentrations was found. Conclusions: PFASs were transferred from mother to foetus, however, with different efficiencies. The concentrations of PFOS, PFOA, PFNA, PFUnDA, and PFDA in foetal organs were much lower than the maternal concentrations. Furthermore, a significant correlation between the exposure duration and all of the evaluated PFASs was found. The health-compromising concentrations of these substances during foetal development are unknown.PostprintPeer reviewe

The fate of germ cells in cryptorchid testis

Cryptorchidism in males constitutes a notable risk factor for both infertility and testicular cancer. Infertility in adulthood is closely linked to the germ cell status in childhood. Furthermore, the significance of germ cell status is important as more than 95% of all reported testicular malignancies are germ cell tumors. The review aims to elucidate the pathogenesis of germ cells in cryptorchid testes concerning their association with infertility and testicular malignancies. Impaired germ cell numbers are evident in cryptorchid testes even during antenatal and neonatal stages. In cryptorchidism there is a rapid decline in germ cell number within the first year of life, partially attributed to physiologic gonocyte apoptosis. Additionally, germ cells fail to differentiate normally during mini-puberty leading to reduced germ cell proliferation and delayed clearance of gonocytes from the seminiferous epithelium. Absence of germ cells in testicular biopsies occurs already 10 months of age and germ cell deterioration progressively worsens with approximately 50% of persisting cryptorchid testes lacking germ cells during puberty. The deficient germ cell maturation and proliferation leads to later infertility. Elevated temperature in the cryptorchid testes and also hormonal deficiency contribute to this phenomenon. Germ cell neoplasia in situ (GCNIS) originating during fetal development may manifest in rare cases associated with disorders of sexual development, chromosomal abnormalities in boys, specific syndromes, and teratomas that include cryptorchidism. In adults, the presence of GCNIS predominantly represents a new histology pattern before invasive germ cell cancer is demonstrated and is neither congenital nor related to abnormal gonocyte transformation