Showing papers in "Reproductive Biology and Endocrinology in 2016"

Genetic, hormonal and metabolic aspects of PCOS: an update.

Abstract: Polycystic ovary syndrome (PCOS) is a complex endocrine disorder affecting 5–10 % of women of reproductive age. It generally manifests with oligo/anovulatory cycles, hirsutism and polycystic ovaries, together with a considerable prevalence of insulin resistance. Although the aetiology of the syndrome is not completely understood yet, PCOS is considered a multifactorial disorder with various genetic, endocrine and environmental abnormalities. Moreover, PCOS patients have a higher risk of metabolic and cardiovascular diseases and their related morbidity, if compared to the general population.

Adenomyosis in infertile women: prevalence and the role of 3D ultrasound as a marker of severity of the disease

Abstract: Adenomyosis is linked to infertility, but the mechanisms behind this relationship are not clearly established. Similarly, the impact of adenomyosis on ART outcome is not fully understood. Our main objective was to use ultrasound imaging to investigate adenomyosis prevalence and severity in a population of infertile women, as well as specifically among women experiencing recurrent miscarriages (RM) or repeated implantation failure (RIF) in ART. Cross-sectional study conducted in 1015 patients undergoing ART from January 2009 to December 2013 and referred for 3D ultrasound to complete study prior to initiating an ART cycle, or after ≥3 IVF failures or ≥2 miscarriages at diagnostic imaging unit at university-affiliated private IVF unit. Adenomyosis was diagnosed in presence of globular uterine configuration, myometrial anterior-posterior asymmetry, heterogeneous myometrial echotexture, poor definition of the endometrial-myometrial interface (junction zone) or subendometrial cysts. Shape of endometrial cavity was classified in three categories: 1.-normal (triangular morphology); 2.- moderate distortion of the triangular aspect and 3.- “pseudo T-shaped” morphology. The prevalence of adenomyosis was 24.4 % (n = 248) [29.7 % (94/316) in women aged ≥40 y.o and 22 % (154/699) in women aged <40 y.o., p = 0.003)]. Its prevalence was higher in those cases of recurrent pregnancy loss [38.2 % (26/68) vs 22.3 % (172/769), p < 0.005] and previous ART failure [34.7 % (107/308) vs 24.4 % (248/1015), p < 0.0001]. The presence of adenomyosis has been shown to be associated to endometriosis [35.1 % (34/97)]. Adenomyosis was diagnosed as a primary finding “de novo” in 80.6 % (n = 200) of the infertile patients. The impact on the uterine cavity was mild, moderate and severe in 63.7, 22.6 and 10.1 % of the cases, respectively. Our results indicate that adenomyosis is a clinical condition with a high prevalence that may affect the reproductive results. The described severity criteria may help future validating studies for better counseling of infertile couples.

Accuracy of preimplantation genetic screening (PGS) is compromised by degree of mosaicism of human embryos

Abstract: To preclude transfer of aneuploid embryos, current preimplantation genetic screening (PGS) usually involves one trophectoderm biopsy at blastocyst stage, assumed to represent embryo ploidy. Whether one such biopsy can correctly assess embryo ploidy has recently, however, been questioned. This descriptive study investigated accuracy of PGS in two ways. Part I: Two infertile couples donated 11 embryos, previously diagnosed as aneuploid and, therefore, destined to be discarded. They were dissected into 37 anonymized specimens, and sent to another national laboratory for repeat analyses to assess (i) inter-laboratory congruity and (ii) intra-embryo congruity of multiple embryo biopsies in a single laboratory. Part II: Reports on human IVF cycle outcomes after transfer of allegedly aneuploid embryos into 8 infertile patients. Only 2/11 (18.2 %) embryos were identically assessed at two PGS laboratories; 4/11 (36.4 %), on repeat analysis were chromosomally normal, 2 mosaic normal/abnormal, and 5/11 (45.5 %) completely differed in reported aneuploidies. In intra-embryo analyses, 5/10 (50 %) differed between biopsy sites. Eight transfers of previously reported aneuploid embryos resulted in 5 chromosomally normal pregnancies, 4 delivered and 1 ongoing. Three patients did not conceive, though 1 among them experienced a chemical pregnancy. Though populations of both study parts are too small to draw statistically adequately powered conclusions on specific degrees of inaccuracy of PGS, here presented results do raise concerns especially about false-positive diagnoses. While inter-laboratory variations may at least partially be explained by different diagnostic platforms utilized, they cannot explain observed intra-embryo variations, suggesting more frequent trophectoderm mosiaicsm than previously reported. Together with recentl published mouse studies of lineages-specific degrees of survival of aneuploid cells in early stage embryos, these results call into question the biological basis of PGS, based on the assumption that a single trophectoderm biopsy can reliably determine embryo ploidy.

Randomized, active-controlled, comparative phase 3 efficacy and safety equivalence trial of Ovaleap® (recombinant human follicle-stimulating hormone) in infertile women using assisted reproduction technology (ART)

Abstract: Pharmacokinetic studies with XM17 (Ovaleap®), a recombinant human follicle-stimulating hormone (r-hFSH, follitropin alfa), have demonstrated good safety and tolerability in healthy women whose endogenous FSH levels were down-regulated with a long agonist protocol. In these studies, Ovaleap® pharmacokinetics were dose-proportional and bioequivalent to the reference follitropin alfa product (Gonal-f®). The objective of the present study is to determine whether Ovaleap® is equivalent to Gonal-f® with respect to the number of oocytes retrieved in infertile but ovulatory women undergoing assisted reproductive technology (ART) therapy. This multinational, multicenter, randomized (1:1), active-controlled, assessor-blind, comparative study included infertile normally gonadotrophic women 18 to 37 years old with a body mass index of 18 to 29 kg/m2 and regular menstrual cycles of 21 to 35 days undergoing ART therapy. During a 5-day fixed-dose phase, women received 150 IU/day of Ovaleap® (n = 153) or Gonal-f® (n = 146), followed by an up to 15-day dose-adaptation phase during which doses could be adjusted every 3 to 5 days, up to a maximum of 450 IU/day. Ovaleap® was to be deemed equivalent to Gonal-f® if the two-sided 0.95 confidence interval (CI) for the difference in the number of oocytes retrieved fell within the equivalence range of ±3 oocytes. Similar numbers of oocytes were retrieved in the 2 treatment groups. The mean ± SD number of oocytes retrieved was 12.2 ± 6.7 in the Ovaleap® group and 12.1 ± 6.7 in the Gonal-f® group (intent-to-treat [ITT] population). Regression analysis estimated a mean difference of 0.03 oocytes between the treatment groups (95 % CI: −0.76-0.82), which was well within the prespecified equivalence range of ±3 oocytes. Ovaleap® and Gonal-f® showed favorable and comparable safety profiles, with no unexpected safety findings. Ovaleap® has shown the same efficacy and safety as Gonal-f® for stimulation of follicular development in infertile women (up to 37 years of age) who are undergoing ART therapy. EudraCT: 2009-017674-20. Current controlled trials: ISRCTN74772901 . Date of trial registration: 19 March 2010.

Sperm motility and morphology changes in rats exposed to cadmium and diazinon.

Abstract: Humans are ubiquitously exposed to multiple environmental contaminants. Consequences of combined action on the reproductive system remain unknown. This study aimed to assess single and joint effects of cadmium and diazinon exposure on sperm quality parameters. Male adult Wistar rats were randomized into 4 groups of ten animals each. Group A was used as a control, animals from group B were exposed to cadmium (30 mg/L), rats from group C were administered with diazinon (40 mg/L), and rats from group D were exposed simultaneously to cadmium (30 mg/L) and diazinon (40 mg/L) via drinking water for 90 days. Sperm morphology and motility were evaluated using a bright field microscope and a computer-assisted semen analysis. The percentage of motile spermatozoa and morphologically normal sperm was markedly reduced in rats from the group B. Rats from the C group showed an increase in velocity parameters, amplitude of lateral head displacement, decrease in beat-cross frequency, and an increase in abnormal sperm morphology. Simultaneous coexposure to cadmium and diazinon increased distance and velocity parameters, and amplitude of lateral head displacement. Reductions were observed in straightness, linearity, wobble, and beat-cross frequency. The decreased normal sperm morphology rates were related to defects of the sperm tail. Exposure to cadmium and diazinon at relatively low doses impairs sperm quality and can reduce male fertility. Cadmium and diazinon caused significant changes on sperm morphology with varying effects on motility patterns. These parameters were significantly higher in the group D as compared to the group C. The findings have important implications for reproductive risk assessment of combined exposures to multiple chemicals.

Effects of hyperandrogenism on metabolic abnormalities in patients with polycystic ovary syndrome: a meta-analysis.

Abstract: The study evaluated the effect of hyperandrogenism (HA) in polycystic ovary syndrome (PCOS) on metabolic parameters. We searched PubMed, EMBASE, Cochrane, Web of Science, Chinese Biomedical Database (CBM), China National Knowledge Infrastructure (CNKI), WanFang data and VIP for clinical observational studies. The study evaluated PCOS patients with or without HA on metabolic parameters was included. Prevalence of metabolic syndrome, indexes of insulin resistance (IR) including homeostasis model assessment IR index (HOMA-IR), incidence of IR, biomarkers of serum lipid metabolism such as total cholesterol (TC), triglyceride (TG), high density lipoprotein (HDL), and low density lipoprotein (LDL). Of 4457 identified trials, 32 observational studies were included for the final analysis comprising 9556 female with PCOS. 6482 cases were having HA, and the others were negative. There were significant differences in the incidence of metabolic syndrome, HOMA-IR, rate of IR, TC level and HDL level between PCOS patients with or without HA, except for LDL level. No significant publication bias was found as P value of Egger’s test was 0.82. HA play an important role in metabolic disorders in PCOS patients. The incidence of metabolic syndrome, IR indexes, and most biomarkers of serum lipid metabolism were significantly different between patients with and without HA.

Risk of adverse pregnancy and perinatal outcomes after high technology infertility treatment: a comprehensive systematic review

Abstract: In the literature, there is growing evidence that subfertile patients who conceived after infertility treatments have an increased risk of pregnancy and perinatal complications and this is particularly true for patients who conceived through use of high technology infertility treatments. Moreover, high technology infertility treatments include many concomitant clinical and biological risk factors. This review aims to summarize in a systematic fashion the current evidence regarding the relative effect of the different procedures for high technology infertility treatments on the risk of adverse pregnancy and perinatal outcome. A literature search up to August 2016 was performed in IBSS, SocINDEX, Institute for Scientific Information, PubMed, Web of Science and Google Scholar and an evidence-based hierarchy was used to determine which articles to include and analyze. Data on prepregnancy maternal factors, low technology interventions, specific procedures for male factor, ovarian tissue/ovary and uterus transplantation, and chromosomal abnormalities and malformations of the offspring were excluded. The available evidences were analyzed assessing the level and the quality of evidence according to the Oxford Centre for Evidence-Based Medicine guidelines and the Grading of Recommendations Assessment, Development, and Evaluation system, respectively. Current review highlights that every single procedure of high technology infertility treatments can play a crucial role in increasing the risk of pregnancy and perinatal complications. Due to the suboptimal level and quality of the current evidence, further well-designed studies are needed.

Which one has a better obstetric and perinatal outcome in singleton pregnancy, IVF/ICSI or FET?: a systematic review and meta-analysis

Abstract: The present study aims to compare which one has a better obstetric and perinatal outcome in singleton pregnancy, frozen embryo transfer (FET) or. in vitro fertilization treatment/intracytoplasmic sperm injection (IVF/ICSI)? MEDLINE, Google Scholar and the Cochrane Library were searched for the obstetric and perinatal outcomes in singleton pregnancy after assisted reproductive technology (ART) from inception until July 2016. Clinical trials, which compared obstetric/perinatal outcomes in singleton pregnancy after FET and IVF/ICSI-ET, were included. The primary outcome was low birth weight, preterm birth, perinatal mortality, still birth, and cesarean section. Thirteen cohort studies with 126,911 women were included, of which 12, 11, 6, 6, 5 studies were used to analyze low birth weight, preterm birth, perinatal mortality, still birth, and cesarean section, respectively. IVF/ICSI is associated with a high risk of preterm birth (OR = 1.14, 95 % CI: 1.02, 1.28) and low birth rate (OR = 1.48, 95 % CI: 1.37, 1.60). There was no significant difference in the risk of the still birth (OR = 1.01, 95 % CI: 0.76, 1.35) and perinatal mortality (OR = 1.11, 95 % CI: 0.85, 1.46) between FET and IVF/ICSI. Singleton pregnancy after FET was associated with higher cesarean section rate compared with IVF/ICSI (OR = 0.85, 95 % CI: 0.80, 0.91). Singleton pregnancy after FET seems to have a better perinatal outcome compared with that after IVF/ICSI. Further randomized controlled trials which adjust for a variety of meaningful confounders are needed in order to draw sound conclusions.

Effects of platelet-rich plasma in a model of bovine endometrial inflammation in vitro.

Abstract: Endometritis reduces fertility and is responsible for major economic losses in beef and dairy industries. The aim of this study was to evaluate an alternative therapy using platelet-rich plasma (PRP). PRP was tested in vivo, after bovine intrauterine administration, and in vitro on endometrial cells. Bovine endometrial cells were cultured until passage (P) 10 with 5 % or 10 % PRP. Effect of PRP on endometrial cell proliferation and on the expression of genes [prostaglandin-endoperoxide synthase 2 (COX2), tumor protein p53 (TP53), oestrogen receptors (ER-α and ER-β), progesterone receptor (PR) and c-Myc] involved in the regulation of oestrus cycle and fetal-maternal interaction were evaluated. Moreover, to evaluate the ability of PRP to counteract inflammation, 10 and 100 ng/ml of bacterial endotoxin lipopolysaccharide (LPS) were used to inflame endometrial cells in vitro for 1, 6, 12, 24 and 48 h. The expression of genes such as interleukin 1β (IL-1β), interleukin-8 (IL-8), inducible nitric oxide synthase (iNOS), prostaglandin-endoperoxide synthase 2 (COX2/PTGS2), and the release of PGE-2, IL-1β and IL-8 were evaluated. In vivo treatment with PRP increased the detection of PR. In vitro, 5 % PRP at passage 5 increased proliferation rate and induced a significant increase in the expression of all studied genes. Furthermore, the results revealed that 10 ng/ml of LPS is the most effective dose to obtain an inflammatory response, and that PRP treatment significantly down regulated the expression of pro-inflammatory genes. This study lays the foundations for the potential treatment of endometritis with PRP in vivo.

Modulation of gonadotrophin induced steroidogenic enzymes in granulosa cells by d-chiroinositol

Abstract: Background d-chiroinositol (DCI) is a inositolphosphoglycan (IPG) involved in several cellular functions that control the glucose metabolism. DCI functions as second messenger in the insulin signaling pathway and it is considered an insulin sensitizer since deficiency in tissue availability of DCI were shown to cause insulin resistance (IR). Polycystic ovary syndrome (PCOS) is a pathological condition that is often accompanied with insulin resistance. DCI can positively affects several aspect of PCOS etiology decreasing the total and free testosterone, lowering blood pressure, improving the glucose metabolism and increasing the ovulation frequency. The purpose of this study was to evaluate the effects of DCI and insulin combined with gonadotrophins namely follicle-stimulating hormone (FSH) and luteinizing hormone (LH) on key steroidogenic enzymes genes regulation, cytochrome P450 family 19 subfamily A member 1 (CYP19A1) and cytochrome P450 side-chain cleavage (P450scc) in primary cultures of human granulosa cells (hGCs). We also investigated whether DCI, being an insulin-sensitizer would be able to counteract the expected stimulator activity of insulin on human granulosa cells (hGCs).

Antioxidants reduce oxidative stress in follicular fluid of aged women undergoing IVF.

Abstract: The status characterized by the imbalance between pro-oxidants and antioxidants molecules, defined as oxidative stress, has been suggested to be involved in the pathogenesis of subfertility in females. This study aims to evaluate the impact of a complete micronutrients supplementation on oxidative stress levels in follicular microenvironment as well as on in vitro fertilization (IVF) outcome. This preliminary study was conducted between January 2014 and July 2015 at the Siena University Hospital Infertility Clinic. Serum and follicular fluid were collected from infertile women aged > 39 years who underwent two in vitro fertilization cycles: in the first cycle they were treated with GnRH-antagonist protocol and gonadotropins for controlled ovarian hyperstimulation, whereas in the second cycle ovarian stimulation protocol was associated to micronutrients supplementation, starting three months earlier. Protein oxidation levels and total antioxidant capacity in serum and in follicular fluid were evaluated in IVF cycles with or without micronutrients supplementation. Differences in IVF outcome parameters were statistically evaluated. Two-dimensional electrophoresis analyses demonstrated that when patients assumed micronutrients before IVF cycles, follicular fluid and serum proteins were protected from oxidative damage. Comparable results were obtained when total antioxidant capacity was measured. Moreover, the mean number of good quality oocytes retrieved when patients received micronutrients supplementation was significantly increased. The additional treatment with micronutrients, starting three months before IVF cycles, protects the follicular microenvironment from oxidative stress, thus increasing the number of good quality oocytes recovered at the pick up.

Biallelic modification of IL2RG leads to severe combined immunodeficiency in pigs

Abstract: Pigs with SCID can be a useful model in regenerative medicine, xenotransplantation, and cancer cell transplantation studies. Utilizing genome editing technologies such as CRISPR/Cas9 system allows us to generate genetically engineered pigs at a higher efficiency. In this study, we report generation and phenotypic characterization of IL2RG knockout female pigs produced through combination of CRISPR/Cas9 system and SCNT. As expected, pigs lacking IL2RG presented SCID phenotype. First, specific CRISPR/Cas9 systems targeting IL2RG were introduced into developing pig embryos then the embryos were transferred into surrogates. A total of six fetuses were obtained from the embryo transfer and fetal fibroblast cell lines were established. Then IL2RG knockout female cells carrying biallelic genetic modification were used as donor cells for SCNT, followed by embryo transfer. Three live cloned female piglets carrying biallelic mutations in IL2RG were produced. All cloned piglets completely lacked thymus and they had a significantly reduced level of mature T, B and NK cells in their blood and spleen. Here, we generated IL2RG knockout female pigs showing phenotypic characterization of SCID. This IL2RG knockout female pigs will be a promising model for biomedical and translational research.

Assisted reproductive technology pregnancy complications are significantly associated with endometriosis severity before conception: a retrospective cohort study.

Abstract: Endometriosis has been shown to be associated with second- to third-trimester pregnancy complications such as preterm birth and placenta previa, but the evidence is inconsistent. We hypothesized that endometriosis severity might affect these inconsistent results. Therefore we aimed to conduct a retrospective cohort study to elucidate whether endometriosis severity is associated with the incidence rates of adverse pregnancy outcomes. The patients who achieved singleton pregnancy by assisted reproductive technology (ART) in our facility between March 2000 and December 2014 (N = 631) were included in this analysis. Among them, 92 women demonstrated surgically proven endometriosis, and 512 women were shown to not have endometriosis as a complication. Among the 92 cases of endometriosis, 10 were classified as revised American Society for Reproductive Medicine (rASRM) stage I and II, 31 cases were rASRM stage III, and 43 cases were rASRM stage IV; in 8 cases, the rASRM stage was unavailable. Logistic regression analysis was performed to calculate odds ratios (OR) and 95 % confidence interval (CI) for the rates of preterm birth, placenta previa, and small for gestational age. OR were adjusted by age, parity and the number of transferred embryos. First we confirmed the frequency of preterm birth and placenta previa were significantly increased in women with endometriosis (preterm birth OR, 2.08; 95 % CI, 1.07–3.89, placenta previa OR, 15.1; 95 % CI, 4.40–61.7), while the frequency of small for gestational age was not. Moreover, we found the frequencies of preterm birth and placenta previa were significantly increased in women with rASRM stage IV endometriosis compared to other two groups: women with rASRM stage I-III endometriosis (preterm birth OR, 7.40; 95 % CI, 1.83–50.3; placenta previa OR, 11.0; 95 % CI, 1.75–216.5) and women without endometriosis (preterm birth adjusted OR, 4.11; 95 % CI, 1.88–8.55; placenta previa adjusted OR, 39.8; 95 % CI, 10.1–189.1). There were no significant difference between women with rASRM I-III endometriosis and women without endometriosis. We found that the frequencies of preterm birth and placenta previa were significantly increased in women with endometriosis, and the severity of endometriosis might have an adverse impact on ART pregnancy.

Assessment of the Access AMH assay as an automated, high-performance replacement for the AMH Generation II manual ELISA.

Abstract: The manual Generation II (Gen II) ELISA method used to measure Anti-Mullerian Hormone (AMH) from Beckman Coulter has recently been superseded by a fully automated AMH immunoassay. The aim of this study was to evaluate the performance of the Access AMH assay and directly compare it to the modified Gen II ELISA method. A secondary aim was to verify that the fertile age-related AMH range previously established using the Gen II ELISA could be used to interpret results from the new automated Access assay. The precision, stability, linearity, measurement range and detection limits were determined using recombinant AMH and patient serum samples. Different diluents and their effects on AMH concentration were compared. A correlation study was performed on patient samples to compare the Access AMH assay to the ELISA method on the Access2 and DxI800 analysers. The fertile AMH range was verified by comparing the 10th, 50th and 90th percentile values from both methods obtained from 489 natural conception pregnant women. The Access AMH assay showed good performance across the measuring range for both intra-assay (CV 1.41–3.30 %) and inter-assay (CV 3.04–5.76 %) precision and acceptable sample stability. Dilution of the high concentration samples with the recommended diluent resulted in a small but significant downward shift in values. The assay was linear over the range of values recommended by the manufacturer, allowing for accurate reporting within the reported range. The two assay types were highly correlated (R2 = 0.9822 and 0.9832 for Access2 and DxI800, respectively), and the differences observed between the Access2 and DxI800 analysers were within clinically acceptable ranges, indicating that the methods are interchangeable. Furthermore, we demonstrated that results from the published reference range for the Gen II ELISA correlate with those from the automated Access AMH assay. Here, we verified the published performance of the Access AMH assay and showed excellent correlation with the Gen II ELISA method. Moreover, we validated this correlation by confirming that the results from a fertile AMH reference range established using the preceding Gen II ELISA are interchangeable with the new automated Access AMH assay.

Abdominal ectopic pregnancy after in vitro fertilization and single embryo transfer: a case report and systematic review.

Abstract: Ectopic pregnancy is the leading cause of maternal morbidity and mortality during the first trimester and the incidence increases dramatically with assisted-reproductive technology (ART), occurring in approximately 1.5–2.1 % of patients undergoing in-vitro fertilization (IVF). Abdominal ectopic pregnancy is a rare yet clinically significant form of ectopic pregnancy due to potentially high maternal morbidity. While risk factors for ectopic pregnancy after IVF have been studied, very little is known about risk factors specific for abdominal ectopic pregnancy. We present a case of a 30 year-old woman who had an abdominal ectopic pregnancy following IVF and elective single embryo transfer, which was diagnosed and managed by laparoscopy. We performed a systematic literature search to identify case reports of abdominal or heterotopic abdominal ectopic pregnancies after IVF. A total of 28 cases were identified. Patients’ ages ranged from 23 to 38 (Mean 33.2, S.D. = 3.2). Infertility causes included tubal factor (46 %), endometriosis (14 %), male factor (14 %), pelvic adhesive disease (7 %), structural/DES exposure (7 %), and unexplained infertility (14 %). A history of ectopic pregnancy was identified in 39 % of cases. A history of tubal surgery was identified in 50 % of cases, 32 % cases having had bilateral salpingectomy. Transfer of two embryos or more (79 %) and fresh embryo transfer (71 %) were reported in the majority of cases. Heterotopic abdominal pregnancy occurred in 46 % of cases while 54 % were abdominal ectopic pregnancies. Our systematic review has revealed several trends in reported cases of abdominal ectopic pregnancy after IVF including tubal factor infertility, history of tubal ectopic and tubal surgery, higher number of embryos transferred, and fresh embryo transfers. These are consistent with known risk factors for ectopic pregnancy following IVF. Further research focusing on more homogenous population may help in better characterizing this rare IVF complication and its risks.

Factors affecting pregnancy outcomes in young women treated with fertility-preserving therapy for well-differentiated endometrial cancer or atypical endometrial hyperplasia

Abstract: Background Patients hoping to preserve their fertility receive conservative treatment with high-dose medroxyprogesterone acetate (MPA) for well-differentiated endometrioid adenocarcinoma (EC) or atypical endometrial hyperplasia (AEH) . Such treatment generally involves frequent intrauterine operations, including dilation and curettage (D&C) and endometrial biopsy (EMB), which could result in endometritis, endometrial thinning, or intrauterine adhesion. In turn, any of these outcomes could adversely affect implantation and pregnancy development. The current study thus aimed to identify factors that might affect pregnancy following conservative treatment by MPA.

MicroRNA-21 and PDCD4 expression during in vitro oocyte maturation in pigs

Abstract: MicroRNA (miRNA) are small non-coding RNA molecules critical for regulating cellular function, and are abundant in the maturing oocyte and developing embryo. MiRNA-21 (MIR21) has been shown to elicit posttranscriptional gene regulation in several tissues associated with rapid cell proliferation in addition to demonstrating anti-apoptotic features through interactions with PDCD4 mRNA and other targets. In many tissues, MIR21 interacts and suppresses PDCD4 due to the strong complementation between MIR21 and the PDCD4 3′UTR. The objective of this project was to examine the relationship between MIR21 and PDCD4 expression in porcine oocytes during in vitro maturation and assess the impact of MIR21 inhibition during oocyte maturation on early embryo development. Additionally, we evaluated the effect of gonadotropins in maturation media and the presence of cumulus cells to determine their ability to contribute to MIR21 abundance in the oocyte during maturation. During in vitro maturation, expression of MIR21 increased approximately 6-fold in the oocyte and 25-fold in the cumulus cell. Temporally associated with this was the reduction of PDCD4 protein abundance in MII arrested oocytes compared with GV stage oocytes, although PDCD4 mRNA was not significantly different during this transition. Neither the presence of cumulus cells nor gonadotropins during in vitro maturation affected MIR21 abundance in those oocytes achieving MII arrest. However, inhibition of MIR21 activity during in vitro maturation using antisense MIR21 suppressed embryo development to the 4–8 cell stage following parthenogenetic activation. MIR21 is differentially expressed in the oocyte during meiotic maturation in the pig and inhibition of MIR21 during this process alters PDCD4 protein abundance suggesting posttranscriptional regulatory events involving MIR21 during oocyte maturation may impact subsequent embryonic development in the pig.

Preimplantation genetic screening- the required RCT that has not yet been carried out.

Abstract: The utilization of trophectoderm biopsy combined with comprehensive chromosome screening (CCS) tests for embryonic aneuploidy was recently suggested to improve IVF outcome, however, not without criticisms. The ongoing discussion on the unrestricted clinical adoption of preimplantation genetic screening (PGS) has called for a proper randomized controlled trial (RCT), aiming to further evaluate the cumulative live birth rates (LBRs) following a single oocyte retrieval, utilizing all fresh and frozen embryos. Since this study seems not to appear for various reasons, we present herewith, the hypothetical required RCT based on the hitherto published literature. After implementing data from the hitherto published literature on blastulation and aneuploidy rates, the rate of mosaicism and technical errors and implantation rates/LBRs of non-PGS day-3 and blastocyst and PGS blastocyst, we could clearly demonstrate the superiority of non-PGS embryo (day-3 and blastocyst) transfer over PGS blastocyst transfer, in terms of cumulative LBR (18.2–50 % vs 7.6–12.6 %, respectively). We therefore believe that until the proper, non-hypothetical RCT on the efficacy of this procedure will appear, PGS should be offered only under study conditions, and with appropriate informed consents.

Different effectiveness of closed embryo culture system with time-lapse imaging (EmbryoScope(TM)) in comparison to standard manual embryology in good and poor prognosis patients: a prospectively randomized pilot study.

Abstract: Previously manual human embryology in many in vitro fertilization (IVF) centers is rapidly being replaced by closed embryo incubation systems with time-lapse imaging. Whether such systems perform comparably to manual embryology in different IVF patient populations has, however, never before been investigated. We, therefore, prospectively compared embryo quality following closed system culture with time-lapse photography (EmbryoScope™) and standard embryology. We performed a two-part prospectively randomized study in IVF (clinical trial # NCT92256309). Part A involved 31 infertile poor prognosis patients prospectively randomized to EmbryoScope™ and standard embryology. Part B involved embryos from 17 egg donor-recipient cycles resulting in large egg/embryo numbers, thus permitting prospectively alternative embryo assignments to EmbryoScope™ and standard embryology. We then compared pregnancy rates and embryo quality on day-3 after fertilization and embryologist time utilized per processed embryo. Part A revealed in poor prognosis patients no differences in day-3 embryo scores, implantation and clinical pregnancy rates between EmbryoScope™ and standard embryology. The EmbryoScope™, however, more than doubled embryology staff time (P < 0.0001). In Part B, embryos grown in the EmbyoScope™ demonstrated significantly poorer day-3 quality (depending on embryo parameter between P = 0.005 and P = 0.01). Suspicion that conical culture dishes of the EmbryoScope™ (EmbryoSlide™) may be the cause was disproven when standard culture dishes demonstrated no outcome difference in standard incubation. Though due to small patient numbers preliminary, this study raises concerns about the mostly uncontrolled introduction of closed incubation systems with time lapse imaging into routine clinical embryology. Appropriately designed and powered prospectively randomized studies appear urgently needed in well-defined patient populations before the uncontrolled utilization of these instruments further expands. NCT02246309 Registered September 18, 2014.

Involvement of oxidative stress in tri-ortho-cresyl phosphate-induced autophagy of mouse Leydig TM3 cells in vitro.

Abstract: As a plasticizer, plastic softener, and flame-retardant, tri-ortho–cresyl phosphate (TOCP) is and has been widely used in industry and reported to have a toxic effect on the male reproductive system in animals besides neurotoxicity and immunotoxicity. We have reported that TOCP inhibits spermatogenesis and induces autophagy of rat spermatogonial stem cells, but it is still unknown whether TOCP induces autophagy of mouse Leydig cells and its potential mechanism. Cell viability was observed by MTT assay. Level of testosterone was measured by radioimmunoassay. Apoptosis was observed by AnnexinV-FITC/PI assay. The contents of LC3, Atg5-Atg12, and Beclin 1 were detected by Western blotting analysis. Autophagosomes were investigated by transmission electron microscopy. The contents of MDA and GSH and the activities of SOD, GSH-PX, total antioxidant status (TAS) and total oxidant status (TOS) were measured by oxidative stress kits. The present study shows that TOCP markedly inhibited viability and testosterone output of mouse Leydig TM3 cells but had no effect on apoptosis. However, TOCP significantly increased both LC3-II and the ratio of LC3-II to LC3-I and the contents of autophagy proteins Atg5 and Beclin 1. Transmission electron microscopy (TEM) showed that TOCP increased autophagic vacuoles of the cytoplasm, indicating that TOCP could induce autophagy of the cells. TOCP significantly induced oxidative stress of mouse Leydig TM3 cells. H2O2 also inhibited viability and induced autophagy of the cells; however, inhibition of oxidative stress by N-acetyl-L-cysteine (NAC) could rescue the inhibition of cell viability and induction of autophagy by TOCP. The results show oxidative stress might be involved in TOCP-induced autophagy of mouse Leydig TM3 cells.

Anti-platelet therapy holds promises in treating adenomyosis: experimental evidence

Abstract: Recently emerging evidence indicates that endometriotic lesions are wounds undergoing repeated tissue injury and repair (ReTIAR), and platelets induce epithelial-mesenchymal transition (EMT), fibroblast-to-myofibroblast transdifferentiation (FMT), leading ultimately to fibrosis. Due to the commonality of cyclic bleeding as in endometriosis, adenomyotic lesions are also wounds that undergo ReTIAR, and we have recently provided evidence corroborating platelet-induced EMT, FMT and fibrogenesis in adenomyosis. This study sought to evaluate the effect of antiplatelet therapy in a mouse model of adenomyosis. Adenomyosis was induced in 57 female ICR mice with neonatal dosing of tamoxifen, while another 12 (group C) were dosed with solvent only, serving as a blank control. Starting from 4 weeks after birth, hotplate test was administrated to all mice every 4 weeks. At the 16th week, all mice with induced adenomyosis were randomly divided into 6 groups: untreated, low- and high-dose Ozagrel, low- and high-dose anti-mouse GPIbα polyclonal IgG antibody to deplete platelets, and isotype-matched inert IgG non-immune antibody. Group C received no treatment. After 3 weeks of treatment, they were hotplate tested again, their uterine horns and brains were harvested, and a blood sample was taken to measure the plasma corticosterone level by ELISA. The left uterine horn was used for immunohistochemistry analysis. The brainstem nucleus raphe magnus (NRM) sections were subjected to immunofluorescence staining for GAD65. The depth of myometrial infiltration and uterine contractility were evaluated. We found that both Ozagrel treatment and platelet depletion dose-dependently suppressed myometrial infiltration, improved generalized hyperalgesia, reduced uterine contractility, and lowered plasma corticosterone levels, improved the expression of some proteins known to be involved in adenomyosis and slowed down the process of fibrogenesis. It also elevated the number of GAD65-expressing neurons in the brainstem NRM, possibly boosting the GABAergic inhibition of pain due to adenomyosis. This study further provides evidence that platelets play important roles in the development of adenomyosis. Anti-platelet treatment is efficacious in suppression of myometrial infiltration, improving generalized hyperalgesia, reducing uterine hyperactivity and systemic corticosterone levels. Collectively, these results demonstrate that anti-platelet therapy seems to be promising for treating adenomyosis.

What affects functional ovarian reserve, thyroid function or thyroid autoimmunity?

Abstract: Thyroid dysfunction is the most common autoimmune endocrine disorder in women of reproductive age, and is associated with menstrual irregularities, anovulation and infertility. Whether it is thyroid function or thyroid autoimmunity that affects functional ovarian reserve (FOR, i.e., the small growing ovarian follicle pool) reflected in anti-Mullerian hormone (AMH) has, however, remained under dispute. We investigated in 225 infertile women whether thyroid function, after adjustment for thyroid autoimmunity, affects FOR within what is considered normal thyroid function (TSH, 0.4–4.5μIU/mL) by assessing AMH levels in reference to TSH levels, stratified for TSH < or ≥3.0μIU/mL. Thyroid autoimmunity was defined by presence of anti-thyroid peroxidase, −thyroglobulin and/or -thyroid receptor antibodies. Mean age of studied women was 38.4 ± 5.0 years; their mean AMH was 1.3 ± 2.0 ng/mL and mean TSH 1.8 ± 0.9 μIU/mL. Thyroid autoimmunity was present in 11.1 % of patients. Women with TSH <3.0μIU/mL presented with significantly higher AMH compared to those with TSH ≥3.0μIU/Ml (P = 0.03). This difference remained significant after adjustment for thyroid autoimmunity as well as age (P = 0.02). Even after adjustment for thyroid autoimmunity and age, TSH <3.0μIU/mL in euthyroid infertility patients is associated with significantly better FOR (higher AMH) than TSH ≥3.0μIU/mL. This observation suggests a direct beneficial effect of lower TSH levels on follicular recruitment, and warrants investigations of thyroxin supplementation in infertile women with TSH levels ≥3.0μIU/mL in attempts to improve FOR.

Expressions of VEGF-A and VEGFR-2 in placentae from GDM pregnancies.

Abstract: Gestational diabetes mellitus (GDM) is one of the most common medical complications of pregnancy, and has important health implications for mother and child. Changes in the fetoplacental vessels may predict those in the vasculature of the developing fetus, as these have been implicated in the pathogenesis of human GDM. This study aimed to determine the differences in the localization and expression level of VEGFA and VEGFR2 between placentas of women with GDM and placentas of normal pregnancies, which is the first step in elucidating the possible roles of VEGFA and VEGFR2 in the altered uteroplacental function resulting from maternal hyperglycaemia and ultimately in the manifestation of GDM. The expressions of VEGFA and VEGFR2 mRNA and protein in 20 samples from each group were analyzed by real-time PCR, immunohistochemistry and Western blot. The placental blood barrier and angiogenesis were observed by the transmission electron microscopy (TEM) in10 GDM samples and ten controls. The expression levels of VEGFA and VEGFR2 mRNA and protein were significantly decreased in the GDM group (P < 0.05 or 0.01). Immunohistochemical analysis showed the reduced expression of VEGFA and VEGFR2 protein in GDM-affected placental tissues, and the degenerative alterations of the terminal villi vascular. The expressions of VEGFA and VEGFR-2 mRNAs and protein were reduced in GDM-affected placental tissues, suggesting that maternal GDM affects the pathophysiological function of placentas.

Relationship between Advanced Glycation End Products and Steroidogenesis in PCOS

Abstract: Women with PCOS have elevated levels of the harmful Advanced Glycation End Products (AGEs), which are highly reactive molecules formed after glycation of lipids and proteins. Additionally, AGEs accumulate in the ovaries of women with PCOS potentially contributing to the well-documented abnormal steroidogenesis and folliculogenesis. A systematic review of articles and abstracts available in PubMed was conducted and presented in a systemic manner. This article reports changes in steroidogenic enzyme activity in granulosa and theca cells in PCOS and PCOS-models. It also described the changes in AGEs and their receptors in the ovaries of women with PCOS and presents the underlying mechanism(s) whereby AGEs could be responsible for the PCOS-related changes in granulosa and theca cell function thus adversely impacting steroidogenesis and follicular development. AGEs are associated with hyperandrogenism in PCOS possibly by altering the activity of various enzymes such as cholesterol side-chain cleavage enzyme cytochrome P450, steroidogenic acute regulatory protein, 17α-hydroxylase, and 3β-hydroxysteroid dehydrogenase. AGEs also affect luteinizing hormone receptor and anti-Mullerian hormone receptor expression as well as their signaling pathways in granulosa cells. A better understanding of how AGEs alter granulosa and theca cell function is likely to contribute meaningfully to a conceptual framework whereby new interventions to prevent and/or treat ovarian dysfunction in PCOS can ultimately be developed.

Aneuploidy analysis in day 7 human blastocysts produced by in vitro fertilization

Abstract: Human embryos produced by in vitro fertilization (IVF) are usually cultured to day 6 after insemination, and good quality of embryos should develop to blastocyst stage at days 5 and 6. However, some embryos develop slowly, thus they may form blastocysts on day 7. Most IVF laboratories culture embryos to day 6 and then discard retarded embryos that do not develop to blastocyst stage. It has been reported that transfer of day 7 blastocysts can yield pregnancy although the rates were very low. In the present study, we evaluated the prevalence of aneuploidy in day 7 human blastocysts and also assessed embryo implantation after transfer of normal euploid blastocysts developed on day 7. Day 7 blastocysts were biopsied and screened for aneuploidy. Embryo implantation was assessed by transferring of euploid blastocysts. A total of 1966 blastocysts from 367 IVF cycles were biopsied and screened for aneuploidy. It was found that 81.5 % of the patients had days 5 and 6 blastocysts and 18.5 % (68) patients had blastocysts developed on day 7, including 15.3 % had days 5–7 blastocysts and 3.3 % had only day 7 blastocysts. A total of 151 day 7 blastocysts, which accounted for 7.7 % of total blastocysts, were analyzed. It was found that 36.7 % of the blastocysts were euploid and 63.3 % had abnormal chromosomes, including aneuploidy and euploid with partial chromosome deletion. The aneuploidy rate was also maternal age dependent and was as high as 91.7 % in patients who were ≥40 years old. During the study period, transfer of day 7 euploid blastocysts in 15 patients resulted in 2 healthy live births. Aneuploidy rates in day 7 human blastocysts produced by IVF are very high. However, good euploid blastocysts have potential to implant and transfer of day 7 euploid blastocysts can result in healthy live birth. It is suggested that day 7 blastocyst culture may be necessary in patients who need aneuploidy screening.

Characterization of exosomal release in bovine endometrial intercaruncular stromal cells

Abstract: Cell-to-cell communication between the blastocyst and endometrium is critical for implantation. In recent years, evidence has emerged from studies in humans and several other animal species that exosomes are secreted from the endometrium and trophoblast cells and may play an important role in cell-to-cell communication maternal-fetal interface during early pregnancy. Exosomes are stable extracellular lipid bilayer vesicles that encapsulate proteins, miRNAs, and mRNAs, with the ability to deliver their cargo to near and distant sites, altering cellular function(s). Furthermore, the exosomal cargo can be altered in response to environmental cues (e.g. hypoxia). The current study aims to develop an in vitro system to evaluate maternal-embryo interactions via exosomes (and exosomal cargo) produced by bovine endometrial stromal cells (ICAR) using hypoxia as a known stimulus associated with the release of exosomes and alterations to biological responses (e.g. cell proliferation). ICAR cells cultured under 8 % O2 or 1 % O2 for 48 h and changes in cell function (i.e. migration, proliferation and apoptosis) were evaluated. Exosome release was determined following the isolation (via differential centrifugation) and characterization of exosomes from ICAR cell-conditioned media. Exosomal proteomic content was evaluated by mass spectrometry. Under hypoxic conditions (i.e. 1 % O2), ICAR cell migration and proliferation was decreased (~20 and ~32 %, respectively) and apoptotic protein caspase-3 activation was increased (∼1.6 fold). Hypoxia increased exosome number by ~3.6 fold compared with culture at 8 % O2. Mass spectrometry analysis identified 128 proteins unique to exosomes of ICAR cultured at 1 % O2 compared with only 46 proteins unique to those of ICAR cultured at 8 % O2. Differential production of proteins associated with specific biological processes and molecular functions were identified, most notably ADAM10, pantetheinase and kininogen 2. In summary, we have shown that a stimulus such as hypoxia can alter both the cellular function and exosome release of ICAR cells. Alterations to exosome release and exosomal content in response to stimuli may play a crucial role in maternal-fetal crosstalk and could also affect placental development.

Effect of nerve growth factor on sperm quality in asthenozoosprmic men during cryopreservation

Abstract: Although routinely used in assisted reproductive technology, human sperm cryopreservation is not an entirely successful procedure. This study determined the effect of nerve growth factor (NGF) supplementation of cryopreservation medium on post-thaw viability, motility, intracellular nitric oxide (NO) concentration, and DNA fragmentation of human spermatozoa in asthenozoospermic men. Semen samples were collected from 25 asthenozoosprmic men and divided into the following groups (n = 5/group): fresh semen (control); frozen-thawed semen without treatment; frozen-thawed semen with NGF treatment (0.5, 1, and 5 ng/ml). Prior to dividing the asthenozoospermic samples, 200 μl of each sample was collected for NGF content assessment by ELISA and then compared with normozoospermic semen samples (25 normozoospermic men). Sperm motility and viability were assessed according to WHO criteria. Furthermore, intracellular nitric oxide and DNA fragmentation were evaluated by Flow Cytometry. NGF content was significantly higher in normozoospermic compared with asthenozoospermic men. Cryopreservation of asthenozoospermic semen samples significantly decreased sperm viability and motility, and increased intracellular nitric oxide concentration and DNA damage (p < 0.01). In asthenozoospermic frozen–thawed samples treated with 0.5 ng/ml exogenous NGF, we observed a significantly increased viability, motility, and decreased DNA fragmentation (p < 0.05), but intracellular nitric oxide concentration was not reduced. The other high doses (1 and 5 ng/ml) had no significant effect on the variables. Supplementation with exogenous NGF could have partial and limited protective effect during cryopreservation of human spermatozoa but further research is needed to evaluate the possible clinical applications.

Biosimilar FSH preparations- are they identical twins or just siblings?

Abstract: As patents expire on innovator products, there is increasing interest in developing biosimilar products globally. Biosimilars are not exact copies and are not considered generic versions of the reference product. They may differ in strength, purity and contain different composition of isoforms and/or various glycosylation profiles, with the consequent alterations in clinical efficacy or safety. Recently 2 new recombinant FSH preparations were introduced to clinical practice following randomized controlled, phase 3 clinical trials. Both, Bemfola and Ovaleap® were referred to the FSH innovator product Gonal-f™ (Follitropin alpha), and were found to yield an equivalent number of oocytes (primary end-point), following a long GnRH agonist suppressive protocol in “ideal” patients, i.e., young, normal responders. However, a closer look at these RCTs reveals a non-significant 4 % difference in clinical and ongoing pregnancy rates, in favor of Gonal f over the biosimilar products, accompanied by half the incidence of OHSS (2.9 vs 5.2 %, respectively). These studies were underpowered with reference to pregnancy rates, Thus, we believe that further comparative studies are needed in additional patient populations, e.g.,older,, poor responders, patients with repeated IVF failures and/or polycystic ovary syndrome, before the universal implementation of biosimilar products for clinical use. Biosimilars are actually a regulatory synonym, facilitating a fast track introduction of a FSH preparation to the COH armamentarium. We therefore recommend against interchanging or substituting innovator and biosimilar agents in clinical practice, and believe that the decision whether to use an innovator or a biosimilar product, should be reserved to the discretion of the treating physician. Furthermore, we believe the time has come that the measurement of the biological activity of FSH in humans should require other methods rather than the Steelman-Pohley assay, such as the determination of dose–response curves in defined populations of women with well-defined outcomes during COH in preparation for ART.

A logistic model to predict early pregnancy loss following in vitro fertilization based on 2601 infertility patients.

Abstract: According to previous studies, even after embryonic cardiac activity is detected, the pregnancy loss rate remains 3–4 %. The objectives of this study were to investigate the differences in ultrasound parameters between a miscarriage group and an ongoing pregnancy group during the 1st trimester and to build a logistic model to predict early pregnancy loss (EPL) after the appearance of embryonic cardiac activity in patients who have undergone in vitro fertilization embryo transfer (IVF-ET) treatment. A total of 2601 patients with early singleton pregnancies with embryonic cardiac activity were retrospectively analyzed after IVF from January 2010 to June 2011. Transvaginal sonography (TVS) was performed at 6 to 10 weeks of gestational age (GA). The mean gestational sac diameter (MSD), crown-rump length (CRL), fetal heart rate (FHR), and yolk sac diameter (YSD) were measured by TVS. A total of 2400 patients had an ongoing pregnancy and an additional 201 (7.7 %) patients miscarried during the first trimester after fetal cardiac activity had been established. The maternal age (MA) and infertility duration were much greater, and the MSD, CRL, and FHR were much lower in the miscarriage group than in the ongoing pregnancy group after IVF (P 5.5). The MA, MSD, CRL, YSD, FHR, infertility duration, and fluid collection around the GS were each correlated with EPL after IVF in infertile patients. A logistic model is a useful tool for predicting EPL after the appearance of embryonic cardiac activity (area under the curve [AUC] = 0.909).

Whether G-CSF administration has beneficial effect on the outcome after assisted reproductive technology? A systematic review and meta-analysis

Abstract: Previous studies have explored the effect of granulocyte colony stimulating factor (G-CSF) administration on the outcome of assisted reproductive technology (ART), and came into controversial conclusions. The present meta-analysis aims to assess whether G-CSF administration has beneficial effect on the outcome after ART. The electronic databases Pubmed, Embase and Google Scholar were searched up to May 2016. Articles that studied the effect of G-CSF administration on the outcome after ART were included in the present meta-analysis. Odds ratio (OR) with 95 % confidence interval (95 % CI) were calculated to assess the effect of G-CSF administration on the outcome after ART. The outcomes of interest were implantation rate (IR) and pregnancy rate (PR). Four cohort studies with 1101 embryos transplantation assessed the effect of G-CSF administration on IR and 6 studies with 621 cycles assessed the role of G-CSF administration in PR. Meta-analysis did not found an increased embryo IR in G-CSF administration cycles [OR 1.59 (95 % CI 0.74–3.41). whereas the PR with G-CSF administration was significantly higher compared with cases without G-CSF administration [OR 2.03 (95 % CI 1.19–3.46)]. Additionally, we found that G-CSF administrated subcutaneously resulted in significantly higher PR [OR 3.12 (95 % CI 1.67–5.81)] and IR [OR 2.82 (95 % CI 1.29–6.15)] compared with control group, whereas G-CSF administrated via local uterine infusion had no beneficial effect on the PR [OR 1.42 (95 % CI 0.91–2.24)] and IR [OR 1.10 (95 % CI 0.76–1.60)] after ART. G-CSF administration may have beneficial effect on clinical pregnancy outcome after ART. Subcutaneous injection may be an optimal route of G-CSF administration. Further cohort studies are required to explore the mechanisms undergone the effect and investigate the best route and dose of G-CSF administration.