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1.  Is it time to shift the attention on early stages embryo development to avoid inconclusive evidence on HPV-related infertility: debate and proposal 
Background
Current evidence about in-vivo effects of HPV cannot definitively clarify the possible negative role of this worldwide common infection in early embryo development. However in-vitro evidence, seems to underline a possible negative effect of HPV in increasing blastocyst apoptosis and in reducing the endometrial implantation of trophoblastic cells. On these bases we believe that a new scientific approach is necessary to better understand the real role of male and female HPV infection in infertility and early pregnancy development.
Methods
English literature review of manuscripts focused on HPV infection and human reproduction was conducted. We performed a critical analysis of evidence and possible bias affecting both in-vivo and in-vitro studies regarding this topic.
Results
The biggest limitation of the in-vivo studies is due to the inappropriate timing of HPV effects evaluation since evidence about in-vitro studies strongly suggests that a large part of HPV negative effects occurs during a very early stage of embryo development. All the efforts of the scientific community to investigate the real role of HPV in human reproduction disorders cannot underestimate the severe BIAS of actual evidence in postulating new hypothesis and research projects which are fundamental to clarify if HPV may be associated with unexplained couples infertility and early miscarriages.
Conclusions
If the relationship between HPV gametes infection and early human reproduction step impairment will be confirmed, the HPV male and couple vaccination may represent a reliable option to improve fertility in some couples affected by infertility actually classified as “idiopathic” but maybe linked to HPV infection.
doi:10.1186/1477-7827-12-48
PMCID: PMC4050410  PMID: 24885125
HPV infection; Human reproduction; Sperm infection; Couple infertility; Early miscarriage; Study BIAS; Embryo development; Blastocyst; Trophoblastic cells
2.  Interplacental uterine expression of genes involved in prostaglandin synthesis during canine pregnancy and at induced prepartum luteolysis/abortion 
Background
In the non-pregnant dog, ovarian cyclicity is independent of a uterine luteolysin. This is in contrast to pregnant animals where a prepartum increase of luteolytic PGF2α occurs, apparently originating in the pregnant uterus. Recently, the placenta as a source of prepartum prostaglandins (PGs) was investigated, indicating fetal trophoblast cells as the likely main source. However, the possible contribution of uterine interplacental tissues to the production of these hormones has not yet been thoroughly examined in the dog.
Methods
Several key factors involved in the production and/or actions of PGs were studied: cyclooxygenase 2 (COX2, PTGS2), PGF2α-synthase (PGFS/AKR1C3), PGE2-synthase (PGES), and the respective receptors FP (PTGFR), EP2 (PTGER2) and EP4 (PGTER4), 15-hydroxyprostaglandin dehydrogenase (HPGD), PG-transporter (PGT, SLCO2A1) and progesterone receptor. Their expression and localization patterns were assessed by Real Time PCR and immunohistology in the interplacental uterine sites from pregnant dogs during the pre-implantation period (days 8–12), post-implantation (days 18–25), mid-gestation (days 35–40) and during antigestagen-induced luteolysis/abortion.
Results
Whereas only low COX2 expression was observed in uterine samples at all the selected time points, expression of PGFS/AKR1C3 strongly increased post-implantation. A gradual increase in PGES-mRNA expression was noted towards mid-gestation. FP-mRNA expression decreased significantly with the progression of pregnancy until mid-gestation. This was associated with clearly detectable expression of HPGD, which did not change significantly over time. The expression of FP and EP2-mRNA decreased significantly over time while EP4-mRNA expression remained unaffected. The antigestagen-treatment led to a significant increase in expression of COX2, PGES, EP2 and PGT (SLCO2A1) mRNA. COX2 was localized predominantly in the myometrium. The expression of PGFS/AKR1C3, which was unchanged, was localized mostly to the surface luminal epithelium. The expression of EP4, PGT and HPGH did not change during treatment, they were co-localized with PGES and EP2 in all uterine compartments.
Conclusions
The data clearly demonstrate the basic capability of the canine pregnant uterus to produce and respond to PGs and suggests their functions both as local regulatory factors involved in the establishment and maintenance of pregnancy, as well as potential contributors to the process of parturition, supporting the myometrial contractility associated with fetal expulsion.
doi:10.1186/1477-7827-12-46
PMCID: PMC4055803  PMID: 24884887
Canine uterus; Prostaglandins
3.  Relationship amongst teratozoospermia, seminal oxidative stress and male infertility 
Background
Spermatozoa morphology is an important and complex characteristic of the fertilization capacity of male germ cells. Morphological abnormalities have been observed to be accompanied by reactive oxygen species (ROS) overproduction and further damage to spermatozoa, ultimately leading to infertility. Therefore, this study aimed to examine the relationship between seminal ROS production and sperm morphology in infertile teratozoospermic patients as well as in healthy men of proven and unproven fertility.
Methods
Semen samples were collected from 79 patients classified as teratozoospermic and 56 healthy donors (control). Standard semen analysis was performed and spermatozoa morphology was assessed according to the WHO 2010 guidelines. Seminal ROS was measured by chemiluminescence assay. Receiver operating characteristic (ROC) curves were generated, and sensitivity, specificity, cutoff value and area under curve (AUC) were determined.
Results
Sperm morphology was significantly poor in the Teratozoospermic Group compared with the 3 Donor Groups (P < 0.05). Significantly higher levels of ROS (RLU/sec/106 sperm) were seen in the Teratozoospermic group (145.4 (41.5; 555.4) compared to the Donor Groups: All Donors (64.8 (21.1; 198.2), Proven Donors (58.8 (14.2; 79.2) and Proven Donors < 2 years (58.8 (14.2; 79.2) (P < 0.05). ROS correlated negatively with sperm concentration in the All Donor group (r = −0.354; P = 0.021) as well as in the Teratozospermic group (r −0.356; P = 0.002). Using ROC analysis, we established the cutoff values for concentration, morphology and ROS.
Conclusions
The incidence of teratozoospermia may be directly related to the overproduction of seminal ROS. Therefore, besides sperm concentration and motility, spermatozoa morphology should receive an equally important consideration in the overall assessment of male fertility.
doi:10.1186/1477-7827-12-45
PMCID: PMC4049374  PMID: 24884815
Spermatozoa; Reactive oxygen species; Morphology; Teratozoospermia; Male infertility
4.  Regulation of ACVR1 and ID2 by cell-secreted exosomes during follicle maturation in the mare 
Background
Ovarian follicle growth and maturation requires extensive communication between follicular somatic cells and oocytes. Recently, intercellular cell communication was described involving cell-secreted vesicles called exosomes (50–150 nm), which contain miRNAs and protein, and have been identified in ovarian follicular fluid. The goal of this study was to identify a possible role of exosomes in follicle maturation.
Methods
Follicle contents were collected from mares at mid-estrous (~35 mm, before induction of follicular maturation) and pre-ovulatory follicles (30–34 h after induction of follicular maturation). A real time PCR screen was conducted to reveal significant differences in the presence of exosomal miRNAs isolated from mid-estrous and pre-ovulatory follicles, and according to bioinformatics analysis these exosomal miRNAs are predicted to target members belonging to the TGFB superfamily, including ACVR1 and ID2. Granulosa cells from pre-ovulatory follicles were cultured and treated with exosomes isolated from follicular fluid. Changes in mRNA and protein were measured by real time PCR and Western blot.
Results
ACVR1 mRNA and protein was detected in granulosa cells at mid-estrous and pre-ovulatory stages, and real time PCR analysis revealed significantly lower levels of ID2 (an ACVR1 target gene) in granulosa cells from pre-ovulatory follicles. Exposure to exosomes from follicular fluid of mid-estrous follicles decreased ID2 levels in granulosa cells. Moreover, exosomes isolated from mid-estrous and pre-ovulatory follicles contain ACVR1 and miR-27b, miR-372, and miR-382 (predicted regulators of ACVR1 and ID2) were capable of altering ID2 levels in pre-ovulatory granulosa cells.
Conclusions
These data indicate that exosomes isolated from follicular fluid can regulate members of the TGFB/BMP signaling pathway in granulosa cells, and possibly play a role in regulating follicle maturation.
doi:10.1186/1477-7827-12-44
PMCID: PMC4045866  PMID: 24884710
Follicular fluid; Ovarian follicle; Exosomes; miRNAs; Equine
5.  Ovulation but not milt production is inhibited in fathead minnows (Pimephales promelas) exposed to a reproductively inhibitory pulp mill effluent 
Background
A 5-day fathead minnow (FHM) spawning assay is used by industry to monitor pulp mill effluent quality, with some mill effluents capable of completely inhibiting spawning. The purpose of this report is to characterize the effect of an inhibitory effluent on egg and milt production in FHM.
Methods
Eight tanks were treated with an inhibitory effluent while eight were kept with clean water. Each tank contained two males and four females as per the 5-day FHM spawning assay used by industry. Females were stripped of ovulated eggs and males of milt in four effluent-exposed and four control tanks. Eggs oviposited in every tank were also counted and checked for fertilization and data analyzed with 2-way ANOVA.
Results
We show that female, but not male, fathead minnow reproductive function is impaired in the 5-day fathead minnow spawning assay used by industry to evaluate pulp mill effluent quality in Canada. Milt production was not changed in the control or exposed males mid-way and at the end of the five day exposure (p > 0.05; n = 8). Total egg production (stripped + oviposited) was impaired (p < 0.05) in fathead minnows exposed to effluent (288 eggs/tank, n = 4 tanks) compared to those in control tanks (753 eggs/tank, n = 4 tanks).
Conclusions
Our results indicate that males are able to detect female signals and prepare appropriately for spawning while in females inhibition of ovulation is occurring somewhere along the hypothalamus-pituitary-gonad reproductive axis. These results suggest female-specific neuroendocrine disruption and provide mechanistic insight into an assay used by industry to assess pulp mill effluent quality.
doi:10.1186/1477-7827-12-43
PMCID: PMC4035717  PMID: 24884628
Fathead minnow; Pulp mill effluent; Reproductive inhibition; 5-day fathead minnow spawning assay; Ovulation; Milt
6.  Increased expression of the pluripotency markers sex-determining region Y-box 2 and Nanog homeobox in ovarian endometriosis 
Background
The precise etiology of endometriosis is not fully understood; the involvement of stem cells theory is a new hypothesis. Related studies mainly focus on stemness-related genes, and pluripotency markers may play a role in the etiology of endometriosis. We aimed to analyze the transcription pluripotency factors sex-determining region Y-box 2 (SOX2), Nanog homeobox (NANOG), and octamer-binding protein 4 (OCT4) in the endometrium of reproductive-age women with and without ovarian endometriosis.
Methods
We recruited 26 women with laparoscopy-diagnosed ovarian endometriosis (endometriosis group) and 16 disease-free women (control group) to the study. Endometrial and endometriotic samples were collected. SOX2, NANOG, and OCT4 expression were analyzed with quantitative real-time polymerase chain reaction, western blotting, and immunohistochemistry.
Results
Compared to the control group, SOX2 mRNA and protein expression was significantly higher in the eutopic endometrium of participants in the endometriosis group. In the endometriosis group, SOX2 and NANOG mRNA and protein expression were significantly increased in ectopic endometrium compared with eutopic endometrium; there was a trend towards lower OCT4 mRNA expression and higher OCT4 protein expression in ectopic endometrium.
Conclusions
The transcription pluripotency factors SOX2 and NANOG were overexpression in ovarian endometriosis, their role in pathogenesis of endometriosis should be further studied.
doi:10.1186/1477-7827-12-42
PMCID: PMC4031377  PMID: 24884521
Endometriosis; Sex-determining region Y-box 2; NANOG; Octamer-binding protein 4
7.  Spontaneous antral follicle formation and metaphase II oocyte from a non-stimulated prepubertal ovarian tissue xenotransplant 
Background
Current strategies in cancer treatment have markedly increased the rates of remission and survival for cancer patients, but are often associated with subsequent sterility. While there are various options available to an adult female depending on the patient’s particular situation, the only realistic option for preserving fertility in prepubertal females is to cryopreserve ovarian tissue. This is the first report of a morphologically mature oocyte collected from non-stimulated prepubertal ovarian tissue xenotransplants.
Methods
Ovarian tissue from a 6 year old patient suffering from nephroblastoma was removed and cryopreserved for fertility preservation. The frozen-thawed ovarian tissue fragments were xenotransplanted to bilaterally oophorectomized severe combined immunodeficiency (SCID) mice to assess follicle development.
Results
Antral follicle formation occurred post-xenotransplantation in a single ovarian fragment without exogenous hormone stimulation. A morphologically maturing oocyte was harvested from these follicles.
Conclusions
Prepubertal human ovarian follicles and oocytes can be matured after xenotransplantation even without exogenous hormone stimulation. These results indicate that tissue collected from prepubertal patients can support fertility in cancer survivors.
doi:10.1186/1477-7827-12-41
PMCID: PMC4036711  PMID: 24886634
Fertility preservation; Ovarian tissue cryopreservation; Prepubertal ovarian tissue; Xenotransplantation; Metaphase II oocyte
8.  Use of antagonists and morpholinos in loss-of-function analyses: estrogen receptor ESR2a mediates the effects of 17alpha-ethinylestradiol on primordial germ cell distribution in zebrafish 
Background
Various chemicals released into the aquatic environment adversely affect the reproductive system of fish, particularly by changing gonad structure and function. 17alpha-ethinylestradiol (EE2) is a potent environmental estrogen that disrupts sexual differentiation and normal reproduction in fish. Previous studies have shown that exposure to endocrine-disrupting chemicals (EDCs) disrupts the migration of primordial germ cells (PGCs) in zebrafish.
Methods
To investigate the effects of EE2 exposure on PGC migration, zebrafish embryos were injected with gfp-nanos mRNA to label PGCs and subsequently exposed to different concentrations of EE2. Typical estrogen receptor antagonist treatment and morpholino knockdown experiments were used to identify functional estrogen receptors that mediate the effects of EE2.
Results
The migration of PGCs was disrupted after exposure to high concentrations of EE2 (1 mirog/L). Loss-of-function analyses were performed for estrogen receptor ESR1, ESR2a, and ESR2b, and only loss of ESR2a resulted in a decreased number of ectopic PGCs following exposure to 1 mirog/L EE2.
Conclusions
EE2 exposure disrupts PGC migration and distribution, and this effect is mediated through the estrogen receptor ESR2a.
doi:10.1186/1477-7827-12-40
PMCID: PMC4038371  PMID: 24886565
17alpha-ethinylestradiol; Primordial germ cells; Estrogen receptor
9.  Rescue of glucocorticoid-programmed adipocyte inflammation by omega-3 fatty acid supplementation in the rat 
Background
Adverse fetal environments predispose offspring to pathologies associated with the metabolic syndrome. Previously we demonstrated that adult offspring of dexamethasone-treated mothers had elevated plasma insulin and pro-inflammatory cytokines, effects prevented by a postnatal diet enriched with omega (n)-3 fatty acids. Here we tested whether prenatal glucocorticoid excess also programmed the adipose tissue phenotype, and whether this outcome is rescued by dietary n-3 fatty acids.
Methods
Offspring of control and dexamethasone-treated mothers (0.75 μg/ml in drinking water, day 13 to term) were cross-fostered to mothers on a standard (Std) or high n-3 (Hn3) diet at birth. Offspring remained on these diets post-weaning, and serum and retroperitoneal fat were obtained at 6 months of age (n = 5-8 per group). Serum was analysed for blood lipids and fatty acid profiles, adipocyte cross sectional area was measured by unbiased stereological analysis and adipose expression of markers of inflammation, glucocorticoid sensitivity and lipid metabolism were determined by RT-qPCR analysis.
Results
Serum total fatty acid levels were elevated (P < 0.01) in male offspring of dexamethasone-treated mothers, an effect prevented by Hn3 consumption. Prenatal dexamethasone also programmed increased adipose expression of Il6, Il1b (both P < 0.05) and Tnfa (P < 0.001) mRNAs regardless of fetal sex, but again this effect was prevented (for Il6 and Il1b) by Hn3 consumption. Offspring of dexamethasone-treated mothers had increased adipose expression of Gr (P = 0.008) and Ppara (P < 0.05) regardless of sex or postnatal diet, while 11bHsd1 was upregulated in males only. The Hn3 diet increased Ppard expression and reduced adipocyte size in all offspring (both P < 0.05) irrespective of prenatal treatment.
Conclusions
Prenatal glucocorticoid exposure programmed increased expression of inflammatory markers and enhanced glucocorticoid sensitivity of adipose tissue. Partial prevention of this phenotype by high n-3 consumption indicates that postnatal dietary manipulations can limit adverse fetal programming effects on adipose tissue.
doi:10.1186/1477-7827-12-39
PMCID: PMC4022445  PMID: 24886466
10.  Early detection and staging of spontaneous embryo resorption by ultrasound biomicroscopy in murine pregnancy 
Background
Embryo resorption is a major problem in human medicine, agricultural animal production and in conservation breeding programs. Underlying mechanisms have been investigated in the well characterised mouse model. However, post mortem studies are limited by the rapid disintegration of embryonic structures. A method to reliably identify embryo resorption in alive animals has not been established yet. In our study we aim to detect embryos undergoing resorption in vivo at the earliest possible stage by ultra-high frequency ultrasound.
Methods
In a longitudinal study, we monitored 30 pregnancies of wild type C57BI/6 mice using ultra-high frequency ultrasound (30-70 MHz), so called ultrasound biomicroscopy (UBM). We compared the sonoembryology of mouse conceptuses under spontaneous resorption and neighbouring healthy conceptuses and correlated the live ultrasound data with the respective histology.
Results
The process of embryo resorption comprised of four stages: first, the conceptus exhibited growth retardation, second, bradycardia and pericardial edema were observed, third, further development ceased and the embryo died, and finally embryo remnants were resorbed by maternal immune cells. In early gestation (day 7 and 8), growth retardation was characterized by a small embryonic cavity. The embryo and its membranes were ill defined or did not develop at all. The echodensity of the embryonic fluid increased and within one to two days, the embryo and its cavity disappeared and was transformed into echodense tissue surrounded by fluid filled caverns. In corresponding histologic preparations, fibrinoid material interspersed with maternal granulocytes and lacunae filled with maternal blood were observed. In later stages (day 9–11) resorption prone embryos were one day behind in their development compared to their normal siblings. The space between Reichert’s membrane and inner yolk sac membrane was enlarged The growth retarded embryos exhibited bradycardia and ultimately cessation of heart beat. Corresponding histology showed apoptotic cells in the embryo while the placenta was still intact. In the subsequent resorption process first the embryo and then its membranes disappeared.
Conclusions
Our results provide a temporal time course of embryo resorption. With this method, animals exhibiting embryo resorption can be targeted, enabling the investigation of underlying mechanisms before the onset of total embryo disintegration.
doi:10.1186/1477-7827-12-38
PMCID: PMC4037759  PMID: 24886361
Embryonic failures; Yolk sac; Reichert’s membrane; Embryo resorption; Ultrasound biomicroscopy; Murine pregnancy; Placenta
11.  Bisphenol a and the female reproductive tract: an overview of recent laboratory evidence and epidemiological studies 
Bisphenol A (BPA) is a high production volume monomer used for making a wide variety of polycarbonate plastics and resins. A large body of evidence links BPA to endocrine disruption in laboratory animals, and a growing number of epidemiological studies support a link with health disorders in humans. The aim of this review is to summarize the recent experimental studies describing the effects and mechanisms of BPA on the female genital tract and to compare them to the current knowledge regarding the impact of BPA impact on female reproductive health. In particular, BPA has been correlated with alterations in hypothalamic-pituitary hormonal production, reduced oocyte quality due to perinatal and adulthood exposure, defective uterine receptivity and the pathogenesis of polycystic ovary syndrome. Researchers have reported conflicting results regarding the effect of BPA on premature puberty and endometriosis development. Experimental studies suggest that BPA’s mechanism of action is related to life stage and that its effect on the female reproductive system may involve agonism with estrogen nuclear receptors as well as other mechanisms (steroid biosynthesis inhibition). Notwithstanding uncertainties and knowledge gaps, the available evidence should be seen as a sufficient grounds to take precautionary actions against excess exposure to BPA.
doi:10.1186/1477-7827-12-37
PMCID: PMC4019948  PMID: 24886252
Bisphenol A; Endocrine disruptors; Fertility; Reproductive health; Genital tract
12.  Kinase insert domain receptor/vascular endothelial growth factor receptor 2 (KDR) genetic variation is associated with ovarian hyperstimulation syndrome 
Background
The objective of this investigation was to determine if kinase insert domain/vascular endothelial growth factor receptor 2 (KDR/VEGFR2) genetic variation was associated with the development of ovarian hyperstimulation syndrome (OHSS) in patients undergoing controlled ovarian hyperstimulation (COH).
Methods
This was a case–control study of 174 patients who underwent controlled ovarian stimulation. Patient blood samples were genotyped for single nucleotide polymorphisms (SNPs) spanning the KDR locus. OHSS development, clinical outcome variables, SNP and haplotype frequencies were compared between control (n = 155) and OHSS (n = 19) groups.
Results
Patients who developed OHSS had significantly higher response markers (estradiol levels of the day of hCG administration, number of follicles developed, number of eggs retrieved) than control patients. When adjusted for age and self-identified race, the rs2305945 G/T genotype was associated (P = 0.027) with a decreased risk (OR = 0.30; 95% CI = 0.10, 0.93) of developing OHSS using an overdominant model. The rs2305945 G/T variant was also associated with decreased COH response (number of follicles, number of eggs retrieved) in an overdominant model. The rs2305948, rs1870378, rs2305945 (C-T-G) haplotype was associated with both decreased COH response and OHSS risk (unadjusted OR = 0.10; 95% CI = 0.01, 0.80, P = 0.031).
Conclusions
The KDR receptor is believed to play a central role OHSS development and is a target for pharmacological prevention of OHSS. These results indicate that genetic variation in the KDR gene may impact individual risk of developing OHSS from COH. In addition, the rs2305948 SNP and C-T-G haplotype might serve as potential biomarkers for poor ovarian response to COH.
doi:10.1186/1477-7827-12-36
PMCID: PMC4024119  PMID: 24886133
Ovarian stimulation; Ovarian hyperstimulation syndrome; OHSS; KDR; VEGFR2; Polymorphism
13.  Systematic analysis of the factors that adversely affect the rate of cell accumulation in mouse embryos during their culture in vitro 
Background
Retarded embryo growth is a pervasive effect of culture in vitro.
Methods
A systematic analysis of the interactions between media design, embryo culture density, oxygen tension, amino acids, trophic ligands and the genetic background of the mouse on embryo growth rates in vitro was performed.
Results
Growth retardation of mouse zygotes was greater in 20% O2 than 5%, a sequential media design was superior to static simple media designs, but the supplementation of simple media with mixed amino acids mitigated this difference. There was a beneficial effect of communal culture in small volumes, and supplementation with a trophic ligand (Paf) further enhanced growth rates. For hybrid strain zygotes (B6CBF1) communal culture in KSOM media supplemented with amino acids, albumin and Paf under 5% O2 resulted in complete rescue of their rate of accumulation of cells and blastocyst formation. Inbred strain (C57BL6/J) zygotes, however, still showed some retardation of development under these conditions. The additional supplementation of media with another trophic ligand (IGF1) showed a further additive beneficial effect on development of inbred strain embryos but they still showed a growth deficit of ~ 23% cell number. The results show that optimising the interactions between a range of culture conditions and media design can rescue hybrid strain embryos from a retarded rate of cell proliferation caused by culture in vitro, but this was incomplete for the B6 strain.
Conclusions
The results indicate that the growth requirement of embryos in vitro varies depending upon their genetic background and provide models for the further genetic analysis of embryo growth.
doi:10.1186/1477-7827-12-35
PMCID: PMC4036297  PMID: 24885989
Reproductive techniques; Zygote; Blastocyst; Embryotrophins; Nutrition; Cell hypoxia
14.  Obesity is associated with increased seminal insulin and leptin alongside reduced fertility parameters in a controlled male cohort 
Background
Obesity appears to be associated with male reproductive dysfunction and infertility, although this has been inconsistent and inconclusive. Insulin and leptin are known mediators and modulators of the hypothalamus-pituitary-testes axis, contributing to the regulation of male reproductive potential and overall wellbeing. These hormones are also present in semen influencing sperm functions. Although abdominal obesity is closely associated with insulin resistance (hyperinsulinaemia), hyperleptinaemia and glucose dysfunction, changes in seminal plasma concentrations of insulin, leptin and glucose in obese males has not previously been investigated.
Methods
This small case controlled study assessed serum and seminal concentrations of insulin, leptin and glucose in obese (BMI > =30; n = 23) and non-obese (BMI < 30; n = 19) males. Following a detailed medical history and examination, participants meeting the inclusion criteria were entered for data analysis. Body parameters such as BMI, waist and hip circumference and the waist hip ratio were measured. Serum and semen samples were collected and assayed for insulin, leptin and glucose. Semen samples also underwent a standard semen analysis, with sperm mitochondrial membrane potential (MMP) and DNA fragmentation (DF).
Results
Obesity was associated with increased serum and seminal insulin and leptin, with no significant difference in seminal glucose. Serum and seminal concentrations of insulin and leptin were positively correlated. Furthermore, obesity was associated with decreased sperm concentration, sperm vitality and increased MMP and DF, with a non-significant impact on motility and morphology.
Conclusions
Hyperinsulinaemia and hyperleptinaemia are associated with increased seminal insulin and leptin concentrations, which may negatively impact male reproductive function in obesity. Insulin was also found to be highly concentrated in the seminal plasma of both groups. This data will contribute to the contradictive information available in the literature on the impact of obesity and male reproduction.
doi:10.1186/1477-7827-12-34
PMCID: PMC4019561  PMID: 24885899
Obesity; Semen; Insulin; Leptin; Glucose; Male fertility
15.  Characterizing semen parameters and their association with reactive oxygen species in infertile men 
Background
A routine semen analysis is a first step in the laboratory evaluation of the infertile male. In addition, other tests such as measurement of reactive oxygen species can provide additional information regarding the etiology of male infertility. The objective of this study was to investigate the association of semen parameters with reactive oxygen species (ROS) in two groups: healthy donors of unproven and proven fertility and infertile men. In addition, we sought to establish an ROS cutoff value in seminal plasma at which a patient may be predicted to be infertile.
Methods
Seminal ejaculates from 318 infertile patients and 56 donors, including those with proven fertility were examined for semen parameters and ROS levels. Correlations were determined between traditional semen parameters and levels of ROS among the study participants. ROS levels were measured using chemiluminescence assay. Receiver operating characteristic curves were obtained to calculate a cutoff value for these tests.
Results
Proven Donors (n = 28) and Proven Donors within the past 2 years (n = 16) showed significantly better semen parameters than All Patients group (n = 318). Significantly lower ROS levels were seen in the two Proven Donor groups compared with All Patients. The cutoff value of ROS in Proven Donors was determined to be 91.9 RLU/s with a specificity of 68.8% and a sensitivity of 93.8%.
Conclusions
Infertile men, irrespective of their clinical diagnoses, have reduced semen parameters and elevated ROS levels compared to proven fertile men who have established a pregnancy recently or in the past. Reactive oxygen species are negatively correlated with traditional semen parameters such as concentration, motility and morphology. Measuring ROS levels in the seminal ejaculates provides clinically-relevant information to clinicians.
doi:10.1186/1477-7827-12-33
PMCID: PMC4047553  PMID: 24885775
Sperm; Motility; Sperm quality; Reactive oxygen species; Infertility
16.  The effect of hormonal estrus induction on maternal effect and apoptosis-related genes expression in porcine cumulus-oocyte complexes 
Background
The effect of hormonal estrus induction on maternal effect (MATER - maternal antigen that embryo requires, ZAR-1 - zygote arrest 1, and BMP15 - bone morphogenetic protein 15) and apoptosis-related genes expression (BCL-2 and BAX) in porcine cumulus-oocyte complexes (COCs) and selected follicular parameters was investigated in this study.
Methods
Gilts were divided into three groups: (I) with natural estrus; (II) stimulated with PMSG/hCG; and (III) with PMSG/hCG + PGF2alpha. Analysis of maternal effect and apoptosis-related transcripts expression in COCs, and progesterone synthesis pathway genes expression (P450scc and 3betaHSD) in granulosa cells was performed by qPCR. BMP15 protein expression in follicular fluid (FF) was analyzed by western blot. Oocyte nuclear maturation was assessed by aceto-orcein staining. Progesterone (P4) and estradiol (E2) concentrations in FF and serum were measured by ELISA. Data were analyzed with the one-way ANOVA and Bonferroni post-test or Kruskal-Wallis test and Dunns post-test.
Results
The highest expression of MATER, ZAR-1, and BMP15 genes was found in COCs recovered from gilts treated with PMSG/hCG when compared to PMSG/hCG + PGF2alpha-stimulated or non-stimulated gilts. Hormonal treatment did not affect the BMP15 protein expression in FF, but increased the expression of genes participating in P4 synthesis in granulosa cells. The higher percentage of immature oocytes was found in PMSG/hCG-treated when compared to the non-stimulated gilts. The expression of BCL-2 and BAX mRNA, and BCL-2/BAX mRNA ratio was significantly higher in COCs derived from PMSG/hCG-treated when compared to PMSG/hCG + PGF2alpha-treated or non-stimulated subjects. The level of P4 in serum was similar in animals from all experimental groups, while its concentration in FF was greater in gilts subjected to PMSG/hCG treatment than in PMSG/hCG + PGF2alpha-stimulated and non-stimulated gilts. The concentration of E2 did not differ in the serum or FF between the control group and the hormonally stimulated groups.
Conclusions
Hormonal induction of estrus affected maternal effect gene transcripts levels in COCs and and oocyte nuclear maturation. The inclusion of PGF2alpha into the stimulation protocol enabled maintaining of physiological concentration of P4 in FF. Additionally, both hormonal treatments seem to be beneficial for apoptosis prevention through increasing BCL-2/BAX transcript ratio.
doi:10.1186/1477-7827-12-32
PMCID: PMC4012087  PMID: 24885667
Maternal effect genes; Pig; Estrus induction; Gonadotropins
17.  Delaying the oocyte maturation trigger by one day leads to a higher metaphase II oocyte yield in IVF/ICSI: a randomised controlled trial 
Background
The negative impact of rising progesterone levels on pregnancy rates is well known, but data on mature oocyte yield are conflicting. We examined whether delaying the oocyte maturation trigger in IVF/ICSI affected the number of mature oocytes and investigated the potential influence of serum progesterone levels in this process.
Methods
Between January 31, 2011, and December 31, 2011, 262 consecutive patients were monitored using ultrasound plus hormonal evaluation. Those with > =3 follicles with a mean diameter of > =18 mm were divided into 2 groups depending on their serum progesterone levels. In cases with a progesterone level < = 1 ng/ml, which was observed in 59 patients, 30-50% of their total number of follicles (only counting those larger than 10 mm) were at least 18 mm in diameter. These patients were randomised into 2 groups: in one group, final oocyte maturation was triggered the same day; for the other, maturation was triggered 24 hours later. Seventy-two patients with progesterone levels > 1 ng/ml were randomised in the same manner, irrespective of the percentage of larger follicles (> = 18 mm). The number of metaphase II oocytes was our primary outcome variable. Because some patients were included more than once, correction for duplicate patients was performed.
Results
In the study arm with low progesterone (<= 1 ng/ml), the mean number of metaphase II oocytes (+/-SD) was 10.29 (+/-6.35) in the group with delayed administration of the oocyte maturation trigger versus 7.64 (+/-3.26) in the control group. After adjusting for age, the mean difference was 2.41 (95% CI: 0.22-4.61; p = 0.031). In the study arm with elevated progesterone (>1 ng/ml), the mean numbers of metaphase II oocytes (+/-SD) were 11.81 (+/-9.91) and 12.03 (+/-7.09) for the delayed and control groups, respectively. After adjusting for PCOS (polycystic ovary syndrome) and female pathology, the mean difference was -0.44 (95% CI: -3.65-2.78; p = 0.79).
Conclusions
Delaying oocyte maturation in patients with low progesterone levels yields greater numbers of mature oocytes.
Trial registration
B67020108975 (Belgian registration) and NCT01980563 (ClinicalTrials.gov).
doi:10.1186/1477-7827-12-31
PMCID: PMC4008411  PMID: 24758641
18.  Effect of addition of FSH, LH and proteasome inhibitor MG132 to in vitro maturation medium on the developmental competence of yak (Bos grunniens) oocytes 
Background
The competence for embryonic development after IVF is low in the yak, therefore, we investigated the effects of supplementation of FSH, LH and the proteasome inhibitor MG132 in IVM media on yak oocyte competence for development after IVF.
Methods
In Experiment 1, yak cumulus-oocyte complexes (COCs) were in vitro matured (IVM) in TCM-199 with 20% fetal calf serum (FCS), 1 microg/mL estradiol-17beta, and different combinations of LH (50 or 100 IU/mL) and FSH (0, 1, 5, 10 microg/mL) at 38.6 degrees C, 5% CO2 in air for 24 h. Matured oocytes were exposed to frozen–thawed, heparin-capacitated yak sperm. Presumptive zygotes were cultured in SOF medium containing 6 mg/ml BSA, 0.5 mg/mL myoinositol, 3% (v/v) essential amino acids, 1% nonessential amino acids and 100 μg/mL L-glutamine (48 h, 38.5 degrees C, 5% CO2, 5% O2, and 90% N2). In Experiment 2, cumulus cells were collected at the end of IVM to determine FSHR and LHR mRNA expression by real-time PCR. In Experiment 3 and 4, COCs were cultured in the presence or absence of the proteasomal inhibitor MG132 from either 0–6 h or 18–24 h after initiation of maturation.
Results
The optimum concentration of FSH and LH in IVM media was 5 microg/mL FSH and 50 IU/mL LH which resulted in the greatest cleavage (79.1%) and blastocyst rates (16.1%). Both FSHR and LHR mRNA were detected in yak cumulus cells after IVM. Treatment with MG132 early in maturation reduced (P < 0.05) cleavage and blastocyst rates. Conversely, treatment with MG132 late in maturation improved (P < 0.05) blastocyst rate. Optimal results with MG132 were achieved at a concentration of 10 microM.
Conclusions
An optimum concentration of FSH and LH in IVM medium, and treatment with MG132 late in maturation can improve yak oocytes competence for development after IVF.
doi:10.1186/1477-7827-12-30
PMCID: PMC3998235  PMID: 24754924
IVF; Yak; FSH; MG132; Early development
19.  Maternal obesity and diabetes may cause DNA methylation alteration in the spermatozoa of offspring in mice 
Background
The adverse effects on offspring of diabetic and/or obese mothers can be passed to the next generation. However, the mechanisms behind this are still unclear. Epigenetics may play a key role during this process.
Methods
To confirm the hypothesis, we investigated the DNA methylation of several imprinted genes in spermatozoa of offspring from diabetic and/or obese mothers utilizing streptozotocin (STZ)- and high-fat-diet (HFD)-induced mouse models.
Results
We found that the DNA methylation of Peg3 was significantly increased in spermatozoa of offspring of obese mothers compared to that in spermatozoa of offspring of normal mothers. The DNA methylation of H19 was significantly higher in spermatozoa of offspring of diabetic mothers than that in spermatozoa of offspring of non-diabetic mothers.
Conclusions
These results indicate that pre-gestational diabetes and/or obesity can alter DNA methylation in offspring spermatozoa.
doi:10.1186/1477-7827-12-29
PMCID: PMC3984639  PMID: 24721882
Spermotozoa; Offspring; Methylation; Maternal diabetes/obesity
20.  The impact of thyroid function on intrauterine insemination outcome - a retrospective analysis 
Background
Hashimoto’s thyroiditis is the most common endocrinopathy in premenopausal women, and is associated with various gynecological problems, including recurrent miscarriage and unexplained infertility. A possible influence of Hashimoto’s thyroiditis on the success of intrauterine insemination seems likely, but has not been evaluated as yet. Therefore, the aim of our study was to retrospectively analyze the impact on intrauterine insemination outcome of thyroid function and markers suggestive for Hashimoto’s thyroiditis.
Methods
Retrospective cohort study in a tertiary care center of 540 women who underwent Intrauterine Insemination. The clinical pregnancy rate was the main outcome parameters. The following possible influencing factors were tested: thyroid-stimulating hormone (TSH); thyroid autoantibodies; age; body mass index; type of sterility (primary/secondary); parity; male factor; presence of PCO syndrome; ovulation induction; ovarian stimulation; and current thyroid medication.
Results
The overall clinical pregnancy rate was 6.9% (37/540). Age, thyroid hormone supplementation for thyroid-stimulating hormone (TSH) levels > 2.5 micro-IU/ml, and ovulation induction with HCG were significantly predictive in the multivariate analysis (p < 0.05) as influencing factors for the pregnancy rate after intrauterine insemination.
Conclusions
Women undergoing intrauterine insemination seem to benefit from a strict thyroid hormone supplementation regimen in order to achieve lower TSH levels.
doi:10.1186/1477-7827-12-28
PMCID: PMC3978130  PMID: 24708845
Intrauterine insemination; Hypothyroidism; Autoimmune thyroiditis; TSH; Target value
21.  Comparison of the biopsy and cytobrush techniques for diagnosis of subclinical endometritis in mares 
Background
Endometritis is a major cause of infertility in the mare. Therefore, the diagnosis of this disease is very important in veterinary practice. The objective of this study was to compare bacteriological and cytological results obtained from the mare uterus using biopsy (EB) and cytobrush (CB) techniques and relating these findings to the presence of polymorphonuclear cells (PMNs) in endometrial tissue as the gold standard for detection of endometritis. In particular, we tested the hypothesis that endometrial cytology and microbiology data obtained from material collected using the EB and CB techniques are similar, so that the CB technique could preferentially be used to detect subclinical endometritis in clinical practice.
Methods
A total of 69 mares suspected of subclinical endometritis because of previous reproductive history and 15 maiden mares were enrolled in this study. Material collected from both EB and CB was smeared on sterile glass slides for cytological examinations and on culture media for microbiological examinations. Bacteriological cultures and cytological samples were classified as negative (no growth or mixed cultures of more than three microorganisms; <2% PMNs) or positive (pure growth of microorganisms; >2% PMNs) for endometritis.
Results
Positive growth was observed in 43% of CB samples and in 54% of EB samples (difference not significant). The growth of β-hemolytic streptococci was always connected with positive cytology. This relationship was not observed for growth of E. coli or for non-pathogenic flora. The sensitivity of bacterial growth and cytology from EB was 0.63 and 0.73 respectively. The sensitivities of bacterial growth and cytology from CB were 0.50 and 0.71 respectively.
Conclusion
Microbiological and cytological results obtained from CB are similar to those obtained from EB and based on these findings the CB technique may be recommended for collection of materials from the mare’s uterus in clinical practice.
doi:10.1186/1477-7827-12-27
PMCID: PMC3986439  PMID: 24708825
Mare; Endometritis; Biopsy; Cytobrush; Cytology
22.  Serum AMH level as a marker of acute and long-term effects of chemotherapy on the ovarian follicular content: a systematic review 
Anti-Müllerian hormone (AMH) is a very sensitive indicator of the ovarian follicular content. Chemotherapeutic agents are notoriously ovariotoxic in that they damage follicles. The aim of this systematic review was to investigate the interest of serum AMH variations in determining the acute and long-term effects of chemotherapy on the ovarian reserve. According to the PRISMA guidelines, searches were conducted on PubMed for all English language articles until December 2013. Fifteen articles that focused on dynamic variations of AMH levels before and after chemotherapy were selected. Cancer patients have significantly lower AMH after chemotherapy than age-matched controls. Longitudinal studies of AMH variations before, during and after chemotherapy provide information about the degree of follicle loss for each patient according to different chemotherapy regimens. Different patterns of AMH levels during the ovarian recovery phase make it possible to discriminate between high and low gonadotoxic chemotherapy protocols. In addition, pretreatment AMH levels are shown to predict the long-term ovarian function after the end of treatment. These results may help to better understand the ovarian toxicity mechanisms of chemotherapy and to predict the degree of the ovarian follicle loss. Therefore, it can be useful for fertility preservation strategies, fertility counseling and future family planning.
doi:10.1186/1477-7827-12-26
PMCID: PMC3987687  PMID: 24666685
AMH; MIS; Cancer; Chemotherapy; Fertility preservation; Follicles; Ovarian toxicity
23.  Leptin receptor signaling inhibits ovarian follicle development and egg laying in chicken hens 
Background
Nutrition intake during growth strongly influences ovarian follicle development and egg laying in chicken hens, yet the underlying endocrine regulatory mechanism is still poorly understood. The relevant research progress is hindered by difficulties in detection of leptin gene and its expression in the chicken. However, a functional leptin receptor (LEPR) is present in the chicken which has been implicated to play a regulatory role in ovarian follicle development and egg laying. The present study targeted LEPR by immunizing against its extracellular domain (ECD), and examined the resultant ovarian follicle development and egg-laying rate in chicken hens.
Methods
Hens that have been immunized four times with chicken LEPR ECD were assessed for their egg laying rate and feed intake, numbers of ovarian follicles, gene expression profiles, serum lipid parameters, as well as STAT3 signaling pathway.
Results
Administrations of cLEPR ECD antigen resulted in marked reductions in laying rate that over time eventually recovered to the levels exhibited by the Control hens. Together with the decrease in egg laying rate, cLEPR-immunized hens also exhibited significant reductions in feed intake, plasma concentrations of glucose, triglyceride, high-density lipoprotein, and low-density lipoprotein. Parallelled by reductions in feed intake, mRNA gene expression levels of AgRP, orexin, and NPY were down regulated, but of POMC, MC4R and lepR up-regulated in Immunized hen hypothalamus. cLEPR-immunization also promoted expressions of apoptotic genes such as caspase3 in theca and fas in granulosa layer, but severely depressed IGF-I expression in both theca and granulosa layers.
Conclusions
Immunization against cLEPR ECD in egg-laying hens generated antibodies that mimic leptin bioactivity by enhancing leptin receptor transduction. This up-regulated apoptotic gene expression in ovarian follicles, negatively regulated the expression of genes that promote follicular development and hormone secretion, leading to follicle atresia and interruption of egg laying. The inhibition of progesterone secretion due to failure of follicle development also lowered feed intake. These results also demonstrate that immunization against cLEPR ECD may be utilized as a tool for studying bio-functions of cLEPR.
doi:10.1186/1477-7827-12-25
PMCID: PMC3976635  PMID: 24650216
Chicken leptin receptor; Extra-cellular domain; Antibody; Ovarian follicule development; Gene expression; Hens
24.  Comparing two different superovulation protocols on ovarian activity and fecal glucocorticoid levels in the brown brocket deer (Mazama gouazoubira) 
Background
Stress is a limiting factor in assisted reproduction in wild animals maintained in captivity. However, the knowledge of assisted reproduction techniques for wild animals is useful for future in situ and ex situ conservation programs. Thus, this study evaluated the ovulation rate, presence of functional corpora lutea and fecal glucocorticoid levels following treatments promoting superovulation in captive brown brocket deer.
Methods
The crossover design used six hinds, allocated to two groups (n = 6): eCG Treatment, CIDR for 8 days, followed by 0.25 mg of EB on day 0, 700 IU of eCG on day 4 following device insertion and 265 mug of PGF2alfa on day 8; and FSH Treatment, CIDR for 7.5 days, followed by 0.25 mg of EB on day 0, 130 mg of FSH in 8 equal doses and 265 mug of PGF2alfa on day 7.5. Induced adrenal activity and treatment efficacy were evaluated by corpora lutea (CL) counts and fecal glucocorticoid and progestin concentration (ng/g feces) analyses for five different phases: Pre, two days before treatment; Early, first four days of treatment; Late, last four days of treatment; Total, entire treatment period; and Post, five days posttreatment.
Results
eCG Treatment resulted in the highest number of CL (P lower than 0.05). There was no significant difference for fecal glucocorticoid concentrations in five different time periods between the treatments; however Pre fecal glucocorticoid concentrations (90.06+/−19.64) were significantly different from Late (200.76+/−26.39) within FSH Treatment. The mean fecal progestin concentration and mean ovulation rate were higher in eCG Treatment (4293.69+/−769.47, 7.0+/−1.8) than in FSH Treatment (1571.26+/−240.28, 2.6+/−0.8) (P lower than or equal to 0.05).
Conclusions
Although the eCG Treatment induced a good superovulatory response, with the formation of functional corpora lutea, we cannot yet affirm that we have established a suitable protocol for induction of SOV in the species M. gouazoubira because approximately 65% of the deer showed premature regression of the corpora lutea. Moreover, multiple FSH applications in FSH Treatment resulted in a low ovulation rate and induced an increase in fecal glucocorticoid levels.
doi:10.1186/1477-7827-12-24
PMCID: PMC3994842  PMID: 24646096
Reproduction biotechniques; Neotropical deer; Fecal glucocorticoids; Fecal progestin
25.  Acne and PCOS are less frequent in women with Mayer-Rokitansky-Küster-Hauser syndrome despite a high rate of hyperandrogenemia: a cross-sectional study 
Background
Acne is a very common skin condition during adolescence and adulthood. Patients with uterovaginal agenesis (Mayer-Rokitansky-Küster-Hauser syndrome, MRKH) treated at the Tübingen University Center for Rare Female Genital Malformations, however, clinically appeared to be less frequently affected by acne. The etiology of MRKH syndrome remains unknown. The only known MRKH-associated mutations are located within the WNT4 gene and lead to an atypical form of MRKH syndrome associated with clinical and biochemical hyperandrogenism. Our study aimed to assess the frequency, severity, and self-evaluation of acne in MRKH patients and to correlate the clinical findings with hormone analyses.
Methods
As part of a cross-sectional longterm follow-up study after laparoscopic assisted creation of a neovagina a questionnaire was sent to 149 MRKH patients aged 16–44 years comprising 26 items concerning prevalence and self-evaluation of acne, and the effects of acne on quality of life. The questionnaire was derived from one used in a former epidemiological study of acne in 4,000 women. Blood for hormone analyses was collected routinely during the clinical visit.
Results
Fully completed, evaluable questionnaires were returned by 69/149 (46%) women. Of these respondents, 42 (60.1%) showed hyperandrogenemia without other clinical signs of virilization but only 17 (24.6%) reported acne (8 (11.6%) had physiological acne and 9 (13.0%) clinical acne) and only 10 (14.5%) reported receiving medical treatment for their acne. Effects of acne on quality of life were minor. Only 4 patients (5.8%) with PCOS were identified, among them one with physiological acne, the other three within the acne-free group.
Conclusions
Although hyperandrogenemia is common, acne is significantly less frequent in women with MRKH than reported in the literature for non-MRKH women, and is seldom treated medically. Patients in this study appeared resistant to acne to some extent, possibly due to the sebaceous glands in the acne regions being less sensitive to androgens compared to the normal population. A WNT4 mutation is unlikely to be the main cause of MRKH syndrome in our hyperandrogenemic patients.
doi:10.1186/1477-7827-12-23
PMCID: PMC4003801  PMID: 24641817
MRKH; Acne; Quality of life; Dermatology life quality index (DLQI); Hyperandrogenemia; WNT4; PCOS

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