Some data suggest that the results of human in vitro fertilization (IVF) may be affected by the site of the uterine cavity where embryos are released. It is not yet clear if there is an optimal range of embryo-fundus distance (EFD) within which embryos should be transferred to optimize IVF outcome.
The present study included 1184 patients undergoing a blind, clinical-touch ET of 1–2 fresh embryos loaded in a soft catheter with a low amount of culture medium. We measured the EFD using transvaginal US performed immediately after ET, with the aim to assess (a) if EFD affects pregnancy and implantation rates, and (b) if an optimal EFD range can be identified.
Despite comparable patients’ clinical characteristics, embryo morphological quality, and endometrial thickness, an EFD between 5 and 15 mm allowed to obtain significantly higher pregnancy and implantation rates than an EFD above 15 mm. The abortion rate was much higher (although not significantly) when EFD was below 5 mm than when it was between 5 and 15 mm. Combined together, these results produced an overall higher ongoing pregnancy rate in the group of patients whose embryos were released between 5 and 15 mm from the fundal endometrial surface.
The site at which embryos are released affects IVF outcome and an optimal EFD range exists; this observations suggest that US-guided ET could be advantageous vs. clinical-touch ET, as it allows to be more accurate in releasing embryos within the optimal EFD range.
Embryo transfer; US-guided embryo transfer; Implantation rate; Pregnancy rate; Abortion rate; Ongoing pregnancy rate
Oocytes may undergo two types of aging. The first is induced by exposure to an aged ovarian microenvironment before being ovulated, known as ‘reproductive or maternal aging’, and the second by either a prolonged stay in the oviduct before fertilization or in vitro aging prior to insemination, known as ‘postovulatory aging’. However, the molecular mechanisms underlying these aging processes remain to be elucidated. As telomere shortening in cultured somatic cells triggers replicative senescence, telomere shortening in oocytes during reproductive and postovulatory aging may predict developmental competence. This study aimed to ascertain the mechanisms underlying altered telomere biology in mouse oocytes during reproductive and postovulatory aging.
We studied Tert expression patterns, telomerase activity, cytosolic reactive oxygen species (ROS) production, and telomere length in fresh oocytes from young versus reproductively-aged female mice retrieved from oviducts at 14 h post-human chorionic gonadotropin (hCG), in vivo or in vitro postovulatory-aged mouse oocytes at 23 h post-hCG. Oocytes were collected from super-ovulated C57BL/6 J mice of 6–8 weeks or 42–48 weeks of age. mRNA and protein expressions of the Tert gene were quantified using real-time quantitative reverse transcriptase polymerase chain reaction (Q-PCR) and immunochemistry. Telomerase activity was measured by a telomeric repeat amplification protocol assay, while telomere length was measured by Q-PCR and quantitative fluorescence in situ hybridization analyses.
The abundance of Tert expression in oocytes significantly decreased during reproductive and postovulatory aging. Immunofluorescent staining clearly demonstrated an altered pattern and intensity of TERT protein expression in oocytes during reproductive aging. Furthermore, relative telomerase activity (RTA) in oocytes from reproductively-aged females was significantly lower than that in oocytes from young females. In contrast, RTA in postovulatory-aged oocytes was similar to that in fresh oocytes. Oocytes from reproductively-aged females and postovulatory-aged oocytes showed higher ROS levels than oocytes from young females. Relative telomere length (RTL) was remarkably shorter in oocytes from reproductively-aged females compared to oocytes from young females. However, postovulatory aging had no significant effect on RTL of oocytes.
Long-term adverse effects of low telomerase activity and increased ROS exposure are likely associated with telomere shortening in oocytes from reproductively-aged female mice.
Reproductive aging; Postovulatory aging; Tert; Oxidative stress; Mouse oocyte; Telomere
Early identification of viable pregnancy is paramount for successful reproduction. Detection of specific signals from pre-implantation viable embryos in normal pregnancy circulation would indicate initiation of embryo-maternal interaction and create a continuum to accurately reflect embryo/fetal well-being post-implantation. Viable mammalian embryos secrete PreImplantation Factor (PIF), a biomarker which plays key, multi-targeted roles to promote implantation, trophoblast invasion and modulate maternal innate and adaptive immunity toward acceptance. Anti-PIF monoclonal antibody (mAb-based chemiluminescent ELISA) accurately detects PIF in singly cultured embryos media and its increased levels correlate with embryo development up to the blastocyst stage. Herein reported that PIF levels (ELISA) in early maternal serum correlate with pregnancy outcome.
Artificially inseminated (AI) blind-coded Angus cattle (N = 21-23) serum samples (day10,15 & 20 post-AI) with known calf birth were blindly tested, using both non-pregnant heifers (N = 30) and steer serum as negative controls. Assay properties and anti-PIF monoclonal antibody specificity were determined by examining linearity, spike and recovery experiments and testing the antibody against 234 different circulating proteins by microarray. Endogenous PIF was detected using <3 kDa filter separation followed by anti-PIF mAb-based affinity chromatography and confirmed by ELISA and HPLC. PIF expression was established in placenta using anti-PIF mAb-based IHC.
PIF detects viable pregnancy at day 10 post-AI with 91.3% sensitivity, reaching 100% by day 20 and correlating with live calf birth. All non-pregnant samples were PIF negative. PIF level in pregnant samples was a stringent 3 + SD higher as compared to heifers and steer sera. Assay is linear and spike and recovery data demonstrates lack of serum interference. Anti-PIF mAb is specific and does not interact with circulating proteins. Anti-PIF based affinity purification demonstrates that endogenous PIF is what ELISA detects. The early bovine placenta expresses PIF in the trophoblast layer.
Data herein documents that PIF is a specific, reliable embryo-derived biomarker conveniently detectable in early maternal circulation. PIF ELISA emerges as practical tool to detect viable early pregnancy from day 20 post-AI.
PreImplantation factor (PIF); Pregnancy BioMarker; ELISA; Live birth; Pregnancy outcome; Monoclonal anti-PIF antibody; Embryo viability
Recurrent spontaneous abortion (RSA) refers to 2 or more consecutive pregnancy losses, and RSA with unknown causes is called unexplained recurrent spontaneous abortion (URSA). Tim-3, a subtype of the T-cell immunoglobulin domain and mucin domain (Tim) protein family, might be an important regulatory molecule that plays a pivotal role in URSA, which might be triggered mostly by Th1/Th2 immune deviation. To understand the etiology and pathogenesis of URSA in Han Chinese women, we investigated the association between polymorphisms of rs10053538 and rs10515746 in the promoter of Tim-3 and the risk of URSA in Han Chinese women.
One hundred and forty-eight women with RSA resulting in still birth were enrolled in the URSA group. We performed tests to rule out congenital reproductive system malformation, reproductive system tumor, endocrine dyscrasia, and chromosome abnormalities. One hundred and fifty-three women with normal pregnancy leading to live birth were selected at random to comprise the control group. All women included in this study were genetically unrelated Han Chinese women. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and allele-specific polymerase chain reaction (AS-PCR) were used to determine polymorphisms of rs10053538 and rs10515746, respectively, in all subjects. PCR products were chosen at random for sequencing.
No significant statistical difference was found between the distribution frequency of the GT + TT genotype and T allele on the rs10053538 locus in the URSA group or the control group (10.1% vs. 11.8%, Chi(2) = 0.205, P = 0.651; 5.1% vs. 6.5%, Chi(2) = 0.592, P = 0.441; respectively). Neither was there a significant difference between the distribution frequency of the GT + TT genotype and T allele on the rs10515746 locus in the groups (6.8% vs. 3.9%, Chi(2)1.201, P = 0.273; 3.4% vs. 2.0%, Chi(2) = 1.169, P = 0.280; respectively).
The present study suggested that these polymorphisms of rs10053538 or rs10515746 in the Tim-3 promoter may not be associated with URSA in Han Chinese women.
Unexplained recurrent spontaneous abortion; Tim-3; Gene polymorphism; Cytokines
Adipose tissue is an active endocrine organ which secretes a wide range of hormones and protein factors, collectively termed adipokines. Adipokines affect appetite and satiety, glucose and lipid metabolism, inflammation and immune functions. The objectives were to evaluate serum concentrations of adipokines (adiponectin, leptin, tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta and IL-6) in lactating dairy cows with postpartum uterine inflammatory conditions (metritis, clinical endometritis or subclinical endometritis) and in cows experiencing loss of body condition, and to assess the relationship of adipokines and body condition loss in the establishment of persistent uterine inflammatory conditions.
Lactating multiparous Holstein cows (N = 40), with body condition scores (BCS) from 2 to 4 (eight cows for each 0.5 score increment) were enrolled. Body condition was monitored for all cows weekly for 7 weeks post calving; cows with uterine inflammatory conditions were also re-evaluated 2 weeks later. Blood samples were collected from 1 week prior to calving to 7 weeks after calving for determination of serum concentrations of adipokines, insulin and insulin like growth factor (IGF)-1.
Cows with metritis or clinical endometritis had higher serum concentrations of adiponectin, leptin, TNF-alpha, IL-1beta and IL-6 compared to normal cows (P < 0.05). Furthermore, serum leptin, TNF-alpha, IL-1beta and IL-6 were higher in cows with subclinical endometritis compared to normal cows (P < 0.05), and insulin and IGF-1 concentrations were lower in cows with metritis or clinical endometritis. Cows with low BCS (2 and 2.5) had significantly higher adiponectin, TNF-alpha, IL-1beta and IL-6 than those with high BCS (3 to 4). Cows with persistent uterine inflammatory conditions had higher adiponectin, leptin TNF-alpha, IL-1beta and IL-6 and insulin compared to normal and spontaneously recovered cows, except for IGF-1 (P < 0.05).
Serum concentrations of adipokines, insulin, and IGF-1 had significant associations with BCS categories (low vs. high) and postpartum uterine inflammatory conditions. Perhaps loss of body condition mediated increases in anti- and pro-inflammatory cytokines, whereas increased pro- and anti-inflammatory cytokines concentrations mediated body condition loss and thereby prolonged persistence of uterine inflammation in dairy cows.
Adipokines; Insulin; IGF-1; Postpartum; Uterine inflammation; Body condition; Dairy cows
Anti-nuclear antibodies (ANA) are suspected of having relevance to adverse reproductive events.
This study aims to investigate the potential effect of ANA on IVF/ICSI outcome and the therapeutic role of prednisone plus low-dose aspirin (P + A) adjuvant treatment in ANA + patients. The first IVF/ICSI cycles without P + A of sixty-six ANA + women were enrolled as the ANA + group, and the 233 first IVF/ICSI cycles of matched ANA- women served as the ANA- group. The ANA + group was divided into the Titre < =1:320 subgroup and the Titre > 1:320 subgroup. Twenty-one ANA + women with adverse outcomes in their first cycles (ANA + cycles without P + A) received P + A adjuvant treatment for three months before the second IVF/ICSI cycle (ANA + cycles with P + A). The clinical characteristics and the IVF/ICSI outcomes were compared, respectively, between 1) the ANA + group and the ANA- group, 2) the Titre < =1:320 subgroup and the Titre > 1:320 subgroup, and 3) the ANA + cycles without P + A and the ANA + cycles with P + A.
No significant differences were observed between each of the two-group pairs in the clinical characteristics. The ANA + group exhibited significantly lower MII oocytes rate, normal fertilisation, pregnancy and implantation rates, as well as remarkably higher abnormal fertilisation and early miscarriage rates. The Titre < =1:320 subgroup’s IVF/ICSI outcomes were as poor as those of the Titre > 1:320 subgroup. After the P + A adjuvant treatment, the number of two pro-nuclei, perfect embryos and available embryos, and the implantation rate increased significantly.
These observations suggest that ANA could exert a detrimental effect on IVF/ICSI outcome that might not be titre-dependent, and P + A adjuvant treatment could be useful for ANA + patients. This hypothesis should be verified in further prospective randomised studies.
Anti-nuclear antibody; ANA; IVF/ICSI outcome; Titre; Prednisone; Aspirin
Letrozole, a non-steroidal aromatase inhibitor, prevents the body from producing its own estrogen. The objectives of the present study were to test the hypotheses that letrozole treatment, initiated prior to selection of the preovulatory dominant follicle, will induce the growth of more than one follicle to a pre-ovulatory size, and will delay ovulation.
Post-pubertal beef heifers were given two luteolytic doses of PGF (12 h apart) and monitored by ultrasonography for ovulation. Five to eight days later, ovarian follicular wave emergence was synchronized by ultrasound-guided transvaginal follicular ablation (Day 0=wave emergence) and a luteolytic dose of PGF was given 60 and 72 h later. On Day 1, heifers were divided randomly into two groups (n=15/group) and an intravaginal device containing 1 g of letrozole or a blank device (control) was inserted. The intravaginal devices were removed on Day 7, or at the time of ovulation, whichever occurred first. Transrectal ultrasonography and blood sample collection were performed daily from the day of ablation to 12 days after subsequent ovulation.
The mean (+/-SEM) interval from device placement to ovulation was longer in letrozole-treated animals compared to controls (6.1+/-0.25 vs 5.1+/-0.26 days, respectively; P<0.01). Single dominant follicles were present in both groups. The day-to-day diameter profiles of the dominant follicles of the ovulatory wave were larger (P<0.05) and the maximum diameters greater in letrozole-treated heifers (14.6+/-0.51 vs 12.4+/-0.53 mm, respectively; P<0.01). The diameter profile of the corpus luteum (CL) that formed after treatment did not differ between groups; however, plasma progesterone concentrations were higher (P<0.01) in heifers treated with letrozole. Estradiol concentrations were reduced following letrozole treatment (P<0.05), although a preovulatory rise of estradiol occurred in both groups.
Administration of letrozole with an intravaginal device during growth of the ovulatory follicle delayed ovulation by 24 h and resulted in the formation of a CL that secreted higher levels of progesterone. A sustained-release intravaginal device may be useful for the development of an aromatase inhibitor-based protocol to control ovulation for herd synchronization and to enhance fertility by increasing circulating progesterone concentrations during the first 7 days post-ovulation in cattle.
Aromatase inhibitor; Letrozole; Ovarian function; Follicle development; Bovine
Both oral contraceptive pills (OCPs) and estradiol (E2) valerate have been used to schedule gonadotropin-releasing hormone (GnRH) antagonist in vitro fertilization (IVF) cycles and, consequently, laboratory activities. However, there are no studies comparing treatment outcomes directly between these two pretreatment methods. This randomized controlled trial was aimed at finding differences in ongoing pregnancy rates between GnRH antagonist IVF cycles scheduled with OCPs or E2 valerate.
Between January and May 2012, one hundred consecutive patients (nonobese, regularly cycling women 18–38 years with normal day 3 hormone levels and <3 previous IVF/ICSI attempts) undergoing IVF with the GnRH antagonist protocol were randomized to either the OCP or E2 pretreatment arms, with no restrictions such as blocking or stratification. Authors involved in data collection and analysis were blinded to group assignment. Fifty patients received OCP (30 μg ethinyl E2/150 μg levonorgestrel) for 12–16 days from day 1 or 2, and stimulation was started 5 days after stopping OCP. Similarly, 50 patients received 4 mg/day oral E2 valerate from day 20 for 5–12 days, until the day before starting stimulation.
Pretreatment with OCP (mean±SD, 14.5±1.7 days) was significantly longer than with E2 (7.8±1.9 days). Stimulation and embryological characteristics were similar. Ongoing pregnancy rates (46.0% vs. 44.0%; risk difference, –2.0% [95% CI –21.2% to 17.3%]), as well as implantation (43.5% vs. 47.4%), clinical pregnancy (50.0% vs. 48.0%), clinical miscarriage (7.1% vs. 7.7%), and live birth (42.0% vs. 40.0%) rates were comparable between groups.
This is the first study to directly compare these two methods of cycle scheduling in GnRH antagonist cycles. Our results fail to show statistically significant differences in ongoing pregnancy rates between pretreatment with OCP and E2 for IVF with the GnRH antagonist protocol. Although the study is limited by its sample size, our results may contribute to a future meta-analysis. An interesting future direction would be to extend our study to women with decreased ovarian reserve, as these are the patients in whom an increase in oocyte yield—due to the hypothetical beneficial effect of steroid pretreatment on follicular synchronization—could more easily be demonstrated.
IVF; GnRH antagonist; Cycle scheduling; Oral contraceptives; Estrogen pretreatment
Endometrial cells, which are shed by retrograde menstruation, may aberrantly express molecules involved in invasion and migration, leading to endometriosis. The aim of this study was to investigate the expression of the laminin gamma 2 chain (LAMC2) in the tissues of women with and without endometriosis.
Endometrial biopsy specimens were collected from healthy volunteers and from endometriosis patients. Biopsy specimens from the corresponding endometriotic lesions were also collected. The expression of laminin gamma 2 chain was evaluated by immunohistochemistry and reverse transcription polymerase chain reaction (RT-PCR).
Endometrial tissue from women with or without endometriosis showed constitutive expression of LAMC2 mRNA throughout the menstrual cycle. A higher mRNA level was observed in ectopic endometrium (Ec) from women with endometriosis compared with eutopic endometrium (Eu) from women with endometriosis. Immunohistochemistry revealed a varied pattern of laminin gamma 2 chain expression, with increased epithelial expression in eutopic endometrium from women with endometriosis compared with those without endometriosis.
The altered expression of laminin gamma 2 chain in eutopic endometrium from women with endometriosis may provide new opportunities for diagnosis and treatment.
Endometrium; Endometriosis; Cell invasion; Adhesion
During the first trimester of pregnancy, trophoblastic E-cadherin expression is down-regulated, thereby allowing extravillous trophoblasts (EVTs) to acquire the potential for migration and invasiveness. The aim of the present study was to investigate the role of OSM on the migration and proliferation of EVT cell line HTR8/SVneo with regard to its effects on the expression of E-cadherin and STAT3 activation.
We investigated the effects of OSM on RNA and protein expression of E-cadherin by real time RT-PCR analyses, western blotting, and indirect immunofluorescence staining in HTR8/SVneo cells, as well as the effects on cell migration and proliferation. The selective signal transducer and activator of transcription (STAT)3 inhibitor, stattic, and STAT3 siRNA were used to investigate STAT3 activation by OSM.
OSM significantly reduced RNA and protein expression of E-cadherin. Indirect immunofluorescence staining of HTR8/SVneo cells also revealed the down-regulation of E-cadherin, compared with the controls. OSM-stimulated cell migration was attenuated by anti-gp130 antibodies. OSM-induced STAT3 phosphorylation, and the down-regulation of E-cadherin by OSM treatment was restored by stattic and STAT3 siRNA. In addition, OSM-stimulated migration and proliferation were significantly suppressed by STAT3 inhibition.
This study suggests that OSM stimulates the migration and proliferation of EVTs during the first trimester of pregnancy through the down-regulation of E-cadherin. In addition, this study suggests that the effects of OSM on migration and proliferation are related to STAT3 activation, which is important in trophoblast invasiveness.
Oncostatin M (OSM); Trophoblast; Migration; Proliferation; E-cadherin; Signal transducer and activator of transcription (STAT)3
When a competent blastocyst stage embryo finds itself in an unreceptive uterus, it delays development. In around one hundred species representing various orders, this delay is known to be reversible, but this phenomenon - termed embryonic diapause (ED) - is not considered a general characteristic of all mammals.
Presentation of the hypothesis
Recently, however, we demonstrated that a non-diapausing species, the sheep, is capable of ED, suggesting the hypothesis that this is in fact an ancestral trait common to all mammals, including humans.
Testing the hypothesis
In spite of the obvious difficulties in testing this idea, we propose a combination of indirect observations on human fertility patients, and direct study of the embryos of non-human primates.
Implications of the hypothesis
Support for our hypothesis would require revision of obstetric interventions routinely performed when a human pregnancy extends beyond the due date.
Embryo; Delayed implantation; Embryonic diapause
Although some studies have reported a potential connection between ovulation induction therapy (OIT) and malignant ovarian diseases, the results have been inconclusive. In the present study, we sought to determine whether women undergoing OIT at our in vitro fertilization (IVF) clinic, especially those with severe ovarian hyperstimulation syndrome (OHSS) and suspicious cytologic findings, were at risk for developing malignant ovarian tumours after treatment.
Patients who underwent OIT at our IVF clinic were enrolled in this study and assessed for any evidence of malignant ovarian tumours. Patients who developed severe OHSS as a result of OIT were treated with a culdocentesis. Cells from the ascitic fluid were cytologically scored for abnormality and malignancy. Peripheral blood samples were obtained from patients with severe OHSS to determine serum levels of the tumour markers (CA-125 and HE4) that were used to calculate the Risk for Ovarian Malignancy Algorithm (ROMA) index.
Follow-up data were available for 1,353 of the 1,587 patients (85%) who underwent OIT at our IVF clinic between January 2006 and December 2012. Twenty-three patients (1.4%) were hospitalized with OHSS. Culdocentesis was performed 16 times in nine patients with severe OHSS (age range, 23–34 years; mean, 27.1 years). Although cytological examination of the ascitic cells of these patients suggested malignant ovarian neoplasia, over the course of the observation period, the ovarian volume gradually decreased and became normal. Subsequent cytological and histological examinations failed to find evidence of any malignant tumours in these nine patients. None of the 1,353 participants who underwent OIT developed any malignant ovarian tumours during the study period. Moreover, none of the 462 patients who were in our ovarian tumour registry were also participants in the IVF program.
The presence of atypical cells in the ascitic fluid of women with severe OHSS does not likely indicate malignancy; therefore, radical surgical intervention is not justified. The risk of malignancy is minimal shortly after OIT. At our centre, OIT has not been associated with any cases of ovarian tumour.
We sought to evaluate the incidence and clinical impact of luteinizing hormone (LH) rises prior to and during gonadotropin-releasing hormone (GnRH) antagonist treatment started on day 5 or 6 of ovarian stimulation with recombinant follicle-stimulating hormone (rFSH).
Pooled data from three trials with the GnRH antagonist ganirelix started on day 5 (n = 961) and from five trials with ganirelix started on day 6 (n = 1135) of ovarian stimulation with rFSH were retrospectively analyzed.
The incidence of LH rises (LH ≥ 10.0 IU/L) prior to ganirelix treatment was 2.3% and 6.6% on ganirelix start days 5 and 6, respectively (P < 0.01). During ganirelix treatment this incidence was 1.2% and 2.3%, respectively (P = 0.06). Women with LH rise on day 5 or 6 had a higher ovarian response with more oocytes recovered, mean ± SD, 12.9 ± 8.5 versus no LH rise, 10.2 ± 6.4 (P < 0.01). In women with and without LH rise prior to ganirelix treatment the ongoing pregnancy rates were similar (26.0% vs 29.9%; odds ratio [OR], 0.89; 95% confidence interval [CI], 0.55-1.44). Women with LH rise during ganirelix treatment had a lower ovarian response with 7.5 ± 6.7 oocytes recovered versus no LH rise, 10.2 ± 6.4 (P = 0.02) and a tendancy for a lower chance of ongoing pregnancy (16.7% vs 29.9%; OR, 0.52; 95% CI, 0.21-1.26).
The incidence of early and late LH rises was low but may be further reduced by initiating ganirelix on stimulation day 5 rather than on day 6. In contrast to women with an early LH rise, women with a late LH rise may have a reduced chance of ongoing pregnancy.
Ovarian stimulation; GnRH antagonist; Serum LH; Ovarian response; Ongoing pregnancy rate
Beta-catenin (CTNNB1), as a key transcriptional regulator in the WNT signal transduction cascade, plays a pivotal role in multiple biological functions such as embryonic development and homeostasis in adults. Although it has been suggested that CTNNB1 is required for gonad development and maintenance of ovarian function in mice, little is known about the expression and functional role of CTNNB1 in gonadal development and differentiation in the chicken reproductive system.
To examine sex-specific, cell-specific and temporal expression of CTNNB1 mRNA and protein during gonadal development to maturation of reproductive organs, we collected left and right gonads apart from mesonephric kidney of chicken embryos on embryonic day (E) 6, E9, E14, E18, as well as testes, oviduct and ovaries from 12-week-old and adult chickens and performed quantitative PCR, in situ hybridization, and immunohistochemical analyses. In addition, localization of Sertoli cell markers such as anti-Müllerian hormone (AMH), estrogen receptor alpha (ESR1), cyclin D1 (CCND1) and N-cadherin (CDH2) during testicular development was evaluated.
Results of the present study showed that CTNNB1 mRNA and protein are expressed predominantly in the seminiferous cords on E6 to E14 in the male embryonic gonad, and are mainly localized to the medullary region of female embryonic gonads from E6 to E9. In addition, CTNNB1 mRNA and protein are abundant in the Sertoli cells in the testes and expressed predominantly in luminal epithelial cells of the oviduct, but not in the ovaries from 12-week-old and adult chickens. Concomitant with CTNNB1, AMH, ESR1, CCND1 and CDH2 were detected predominantly in the seminiferous cord of the medullary region of male gonads at E9 (after sex determination) and then maintained or decreased until hatching. Interestingly, AMH, ESR1, CCND1 and CDH2 were located in seminiferous tubules of the testes from 12-weeks-old chickens and ESR1, CCND1 and CDH2 were expressed predominantly in the Sertoli cells within seminiferous tubules of adult testes.
Collectively, these results revealed that CTNNB1 is present in gonads of both sexes during embryonic development and it may play essential roles in differentiation of Sertoli cells during formation of seminiferous tubules during development of the testes.
CTNNB1; Chicken; Gonad; Testis; Sertoli cell
Granulosa cells play a key role in folliculogenesis and female reproduction. Our previous study demonstrated that water channel aquaporin-8 (AQP8) is expressed in mouse follicular granulosa cells and is an important determinant of granulosa cell apoptosis and follicular maturation. More roles of AQP8 in folliculogenesis remain to be determined.
The present study reports the increased occurrence of multi-oocyte follicles (MOFs) in ovaries of AQP8 knockout mice. The MOFs in AQP8-deficient ovaries contained two or three oocytes, and distributed at various follicle stages including primary (12.5%), secondary (50%), antral (18.8%) and atretic (18.8%) follicles in 5-week ovaries. The MOF is occasionally seen in wild-type ovary only in primary and secondary follicles. The number of MOFs in AQP8-deficient ovary reduced with age (26.7 +/− 5.2 per ovary at 5 weeks old, 14 +/− 5.5 at 10 weeks old, and 3.3 +/− 5.1 at 20 weeks old). mRNA expression of AQP5, AQP7, AQP8, AQP11 and AQP12 was detected in neonatal mouse ovaries and in granulosa cells in 4 week old mouse ovaries. The expression of AQP7, AQP11 and AQP12 mRNAs are decreased significantly in neonatal AQP8-deficient ovaries, whereas AQP5 mRNA expression remains unchanged.
The emergence of MOFs is associated with AQP8 deficiency. The study suggested the involvement of AQP8 in the formation of follicles and provided new insight into the molecular mechanisms of folliculogenesis.
Aquaporin-8; Multi-oocyte follicle; Folliculogenesis; Formation of follicle
Oocyte-specific genes play critical roles in oogenesis, folliculogenesis and early embryonic development. The objectives of this study were to characterize the expression of a novel oocyte-specific gene encoding an F-box protein during ovarian development in rainbow trout, and identify its potential interacting partners in rainbow trout oocytes.
Through analysis of expressed sequence tags (ESTs) from a rainbow trout oocyte cDNA library, a novel transcript represented by ESTs only from the oocyte library was identified. The complete cDNA sequence for the novel gene (named fbxoo) was obtained by assembling sequences from an EST clone and a 5′RACE product. The expression and localization of fbxoo mRNA and protein in ovaries of different developmental stages were analyzed by quantitative real time PCR, immunoblotting, in situ hybridization and immunohistochemistry. Identification of Fbxoo binding proteins was performed by yeast two-hybrid screening.
fbxoo mRNA is specifically expressed in mature oocytes as revealed by tissue distribution analysis. The fbxoo cDNA sequence is 1,996 bp in length containing an open reading frame, which encodes a predicted protein of 514 amino acids. The novel protein sequence does not match any known protein sequences in the NCBI database. However, a search of the Pfam protein database revealed that the protein contains an F-box motif at the N-terminus, indicating that Fbxoo is a new member of the F-box protein family. The expression of fbxoo mRNA and protein is high in ovaries at early pre-vitellogenesis stage, and both fbxoo mRNA and protein are predominantly expressed in early pre-vitellogenic oocytes. Several proteins including tissue inhibitor of metalloproteinase 2 (Timp2) were identified as potential Fbxoo protein binding partners.
Results suggest that the novel oocyte-specific F-box protein may play an important role in early oocyte development by regulating other critical proteins involved in oogenesis in rainbow trout.
F-Box protein; Oocyte; Vitellogenesis; Rainbow trout
In vitro fertilization involves high dosage gonadotropin stimulation, which apparently has some negative impact on follicular endocrine function. As chorionic gonadotropin stimulation has been shown to increase the blood-follicular permeability in animal models, this raises the question if such an effect also applies to gonadotropins in humans, possibly affecting the endocrine follicular milieu.
Follicular fluid and serum were collected at the time of follicular aspiration in in vitro fertilisation without (Natural cycle IVF, n = 24) and with (conventional gonadotropin stimulated IVF, n = 31) gonadotropin stimulation. The concentration of the extra-ovarian hormones prolactin and cortisol were analysed by immunoassays.
Median serum prolactin and cortisol concentrations were 12.3 ng/mL and 399 nmol/L without versus 32.2 ng/mL and 623 nmol/L with gonadotropin stimulation. The corresponding concentrations in follicular fluid were 20.6 ng/mL and 445 nmol/L versus 28.8 ng/ml and 456 nmol/L for prolactin and cortisol. As a consequence, mean follicular fluid:serum ratios were significantly reduced under gonadotropin stimulation (prolactin p = 0.0138, cortisol p = 0.0001). As an enhanced blood-follicular permeability and transportation, induced by gonadotropin stimulation, would result in increased instead of decreased follicular fluid:serum ratios as found in this study, it can be assumed that this does not affect extra-ovarian protein and steroid hormones as illustrated by prolactin and cortisol.
The model of serum follicular fluid:serum ratio of hormones, produced outside the ovaries, did not reveal a gonadotropin induced increased blood-follicular transportation capacity. Therefore it can be assumed that the effect of gonadotropins on follicular endocrine function is not due to an increased ovarian permeability of extra-ovarian hormones.
Follicular fluid; Natural Cycle In vitro Fertilisation; Permeability; Gonadotropin; Prolactin; Cortisol
Seminal plasma serves as a natural reservoir of antioxidants. It helps to remove excessive formation of reactive oxygen species (ROS) and consequently, reduce oxidative stress. Proteomic profiling of seminal plasma proteins is important to understand the molecular mechanisms underlying oxidative stress and sperm dysfunction in infertile men.
This prospective study consisted of 52 subjects: 32 infertile men and 20 healthy donors. Once semen and oxidative stress parameters were assessed (ROS, antioxidant concentration and DNA damage), the subjects were categorized into ROS positive (ROS+) or ROS negative (ROS-). Seminal plasma from each group was pooled and subjected to proteomics analysis. In-solution digestion and protein identification with liquid chromatography tandem mass spectrometry (LC-MS/MS), followed by bioinformatics analyses was used to identify and characterize potential biomarker proteins.
A total of 14 proteins were identified in this analysis with 7 of these common and unique proteins were identified in both the ROS+ and ROS- groups through MASCOT and SEQUEST analyses, respectively. Prolactin-induced protein was found to be more abundantly present in men with increased levels of ROS. Gene ontology annotations showed extracellular distribution of proteins with a major role in antioxidative activity and regulatory processes.
We have identified proteins that help protect against oxidative stress and are uniquely present in the seminal plasma of the ROS- men. Men exhibiting high levels of ROS in their seminal ejaculate are likely to exhibit proteins that are either downregulated or oxidatively modified, and these could potentially contribute to male infertility.
Seminal plasma; Male infertility; Varicocele; Reactive oxygen species; Oxidative stress; Proteomics; Mass spectroscopy; Differentially expressed proteins; Gene ontology
Although most pregnancies after IVF result in normal healthy outcomes, an increased risk for a number of obstetric and neonatal complications, compared to naturally conceived pregnancies, has been reported. While there are many studies that compare pregnancies after assisted reproductive techniques with spontaneously conceived pregnancies, fewer data are available that evaluate the differences between IVF and ICSI-conceived pregnancies. The aim of our present study was, therefore, to compare obstetric and perinatal outcomes in pregnancies conceived after in vitro fertilization (IVF) versus intracytoplasmatic sperm injection (ICSI).
Three-hundred thirty four women who had become pregnant after an IVF or ICSI procedure resulted in a total of 530 children referred between 2003 und 2009 to the Department of Obstetrics and Gynecology of the Medical University of Vienna, a tertiary care center, and were included in this retrospective cohort study. We assessed maternal and fetal parameters in both groups (IVF and ICSI). The main study outcomes were preterm delivery, the need for neonatal intensive care, and congenital malformations. Moreover, we compared the course of pregnancy between both groups and the occurrence of complications that led to maternal hospitalization during pregnancy.
There were 80 children conceived via ICSI and 450 children conceived via IVF.
Mean gestational age was significantly lower in the ICSI group (p = 0.001). After ICSI, the birth weight (p = 0.008) and the mean APGAR values after 1 minute and after 10 minutes were lower compared to that of the IVF group (p = 0.016 and p = 0.047, respectively). Moreover, ICSI-conceived children had to be hospitalized more often at a neonatal intensive care unit (p = 0.004). There was no difference in pH of the umbilical artery or in major congenital malformations between the two groups. Pregnancy complications (i.e., premature rupture of membranes, cervical insufficiency, and premature uterine contractions) and the need for maternal hospitalization during pregnancy were found significantly more often after IVF (p = 0.0016 and p = 0.0095, respectively), compared to the ICSI group.
When comparing IVF versus ICSI-conceived pregnancies at a tertiary care center, we found the course of pregnancy to be more complicated after IVF, whereas the primary fetal outcome seemed to be better in this group than after ICSI treatment.
IVF; ICSI; Pregnancy course; Obstetric outcome; Perinatal outcome
Declining numbers of wild giant anteaters highlight the importance of sustainable captive populations. Unfortunately, captive reproductive management is limited by the lack of external physical indicators of female reproductive status and the aggressive behavior of males. We examined the endocrinology of the estrous cycle and pregnancy, and whether delayed implantation is a gestational strategy for giant anteaters as described for other xenarthrans.
Feces were collected from seven captive females 3–5 times weekly and mating was recorded. Concentrations of estrogen (estrone–glucuronide, E1, and estradiol–17β, E2), progestagen (20–oxo–progestagens, P4), and glucocorticoid (GC) metabolites were examined in fecal extracts by enzyme immunoassay.
Estrous cycles for nulliparous females (6 cycles, n = 2) compared to the multiparous female (6 cycles, n = 1) were shorter (47.3 +/− 4.3 days versus 62.5 +/− 2.6 days) with relatively lower luteal phase concentrations of P4 (49.4 +/− 2.9 ng/g versus 136.8 +/− 1.8 ng/g). The four remaining females had unclear ovarian activity: two females exhibited apparent luteal activity but unclear fluctuations in estrogens, while the other two females had parallel fecal P4 and estrogens concentrations. Pregnancy ranged 171–183 days with females returning to estrus post–partum as early as 60 days (n = 3, 1.8-4 years of age at mating). Delayed implantation was indicated by a biphasic elevation in fecal P4 metabolites: the initial 4–fold increase occurred for 81–105 days and was followed by a 26–fold secondary rise in P4 metabolites lasting 66–94 days prior to parturition. Fecal GC was correlated with fecal estrogens and greatest during estrus, late pregnancy, and six days prior to parturition (estrous cycle GC, 14.4-62.8 ng/g; pregnancy GC, 13.6-232.7 ng/g).
Estrous cycles of giant anteaters occurred year–round, but were shorter and more intermittent in younger nulliparous animals compared to a multiparous female. A pronounced elevation in fecal P4, estrogen, and GC occurred during late gestation after an initial post-mating delay providing evidence for delayed implantation. Adrenocorticoid activity indicated impending parturition. Differences in estrous cycle characteristics with age and the protracted but variable gestation length must be considered to improve reproductive success and neonatal survival in giant anteaters.
Enzyme immunoassays; Fecal steroid hormones; Gestation; Non–invasive monitoring; Xenarthra
Hypothesizing that redundant functional ovarian reserve (FOR) at young ages may clinically obfuscate prematurely diminished FOR (PDFOR), we investigated in young oocyte donors genotypes and sub-genotypes of the FMR1 gene, in prior studies associated with specific ovarian aging patterns, and determined whether they already at such young age were associated with variations in ovarian reserve (OR). We also investigated racial as well as FMR1 associations with menarcheal age in these donors.
In a cohort study we investigated 157 oocyte donor candidates and, based on the 95% CI of AMH, divided them into normal age-specific (AMH greater or equal to 2.1 ng/mL; n = 121) and PDFOR (AMH < 2.1 ng/mL; n = 36). We then assessed associations between numbers of trinucleotide repeat (CGGn) on the FMR1 gene and FOR (based on anti-Müllerian hormone, AMH).
FMR1 did not associate with AMH overall. Amongst 36 donors with PDFOR, 17 (42%) presented with at least one low (CGGn < 26 ) allele. Remaining donors with normal FOR presented with significantly more CGGn greater or equal to 26 (73.6% vs. 26.4%; P = 0.024) and higher AMH (P = 0.012). This finding was mostly the consequence of interaction between FMR1 (CGGn < 26 vs. CGGn greater or equal to 26) and race (P = 0.013), with Asians most responsible (P = 0.009). Menarcheal age was in donors with normal FOR neither associated with race nor with FMR1 status. In donors with PDFOR race was statistically associated with CGGn (P = 0.018), an association primarily based on significantly delayed age of menarche in African donors with CGGn < 26 in comparison to African donors with CGGn greater or equal to 26 (P = 0.019), and Caucasian (P = 0.017) and Asian donors (P = 0.025) with CGGn < 26.
CGGn on FMR1 already at young ages affects FOR, but is clinically apparent only in cases of PDFOR. Screening for low FMR1 CGGn < 26 at young age, thus, appears predictive of later PDFOR.
Fragile X gene; FMR1 gene; Ovarian reserve; Anti-müllerian hormone (AMH); Oocyte donor
Spermatozoa are extremely vulnerable to oxidative stress caused by the unbalance between concentrations of reactive oxygen species and antioxidant scavenging systems present inside the male reproductive tract. In spite of a large number of clinical studies that claimed the beneficial effects of antioxidant oral administration on sperm physiology and fertility, only a few studies were addressed to evaluate their effects on spermatozoa in vitro. Main aims of the present study were to assess the influence of zinc, D-aspartate and coenzyme Q10, included in the dietary supplement Genadis (Merck Serono), on human sperm motility, DNA fragmentation and lipid peroxidation.
Semen samples, obtained from forty-four patients (23–30 years of age) were enrolled in this study, twenty-four were normospermic and twenty patients were oligospermic. Semen samples were analysed for sperm progressive motility and kinetics through computer assisted analysis, DNA fragmentation and lipid peroxidation.
Main results showed that in both normo and oligospermic samples, total and progressive sperm motility is maintained by in vitro treatment with zinc, D-aspartate and coenzyme Q10, whereas a significant decrease of these parameters occurs in parallel samples incubated in medium alone. Zinc, D-aspartate and coenzyme Q10 also prevented the decrease of sperm kinetics but such an effect was highly significant only in oligospermic samples. Moreover, they also protected spermatozoa by the increase of DNA fragmentation and lipid peroxidation.
Zinc, D-aspartate and coenzyme Q10 exert a direct protective effect on human spermatozoa preventing the decrease of motility and the increase of DNA fragmentation and lipid peroxidation during in vitro culture.
Spermatozoa; Antioxidants; Motility; Lipid peroxidation; DNA fragmentation
Obesity is becoming an increasing problem in obstetric practice; it has led to an increase in the risk of caesarean delivery, prolonged pregnancy and dysfunctional labour. It has been postulated that many of these problems are as a result of abnormal myometrial contractility. The RhoA/Rho kinase pathway is involved in calcium sensitisation in the myometrium during labour and contributes to the phosphorylation of myosin phosphatase and thus continued myosin light chain activity, during uterine contractility. The aim of this study therefore, was to investigate the effect of obesity on the expression of various components of the RhoA/ROCK pathway in human myometrium at term pregnancy.
Protein was isolated from myometrial biopsies obtained at elective caesarean section, at term pregnancy from obese women and from those with a normal body mass index. Western blotting was performed using specific primary antibodies to RhoA/ Rho kinase associated proteins.
The protein expression of p160 ROCK-1 was significantly decreased (P < 0.001) in the myometrium from women in the obese cohort (n = 22) at term pregnancy, compared to women of those of normal body mass index (n = 15). No alteration in expression of the other proteins investigated was noted.
The significant decrease in p160 ROCK-1 protein expression observed in the myometrium of obese women at late gestation may contribute to an inhibitory effect on contractility at labour, due to its contribution to calcium sensitisation and possibly other signalling pathways. These findings are relevant to the concept of compromised myometrial function in obese parturients.
p160 ROCK 1 protein; Myometrium; Obesity; Term pregnancy
The eutopic endometrium of women with endometriosis, compared with disease-free individuals, contains certain molecular alterations, including the differential expression of microRNA (miRNA). The aim of the study was to compare the expression of the most relevant miRNAs in the eutopic endometrium of women with and without ovarian endometriosis.
A total of 46 regularly menstruating patients, 21 patients with ovarian endometriosis and 25 controls, underwent surgery in the proliferative phase of the cycle. The eutopic endometrium was collected through aspirating biopsy prior to laparoscopy. Only patients with advanced (stage III and IV) histopathologically confirmed ovarian endometriosis were included. TaqMan MicroRNA Array Cards were applied to examine the expression of 667 human miRNAs in 10 patients with endometriosis and 10 controls. Custom-made, low-density real-time PCR arrays were used to confirm the expression of 15 selected molecules in 21 endometriosis patients and 25 disease-free individuals.
Of 667 miRNAs, 2 were highly likely to be upregulated and 13 were downregulated in the eutopic endometrium of patients with endometriosis compared with the controls. Validation using real-time PCR showed that hsa-miR-483-5p (p = 0.012) and hsa-miR-629* (p = 0.02) are significantly downregulated in patients with endometriosis.
Changes in the expression of select miRNAs might lead to or be a consequence of an early defect in the physiological activity of the proliferative endometrium, ultimately resulting in the overgrowth of this tissue outside the uterus.
Endometriosis; Eutopic endometrium; MiRNAs