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1.  Local uterine Ang-(1–7) infusion augments the expression of cannabinoid receptors and differentially alters endocannabinoid metabolizing enzymes in the decidualized uterus of pseudopregnant rats 
Endocannabinoids (ECs) are important contributors to implantation and decidualization and are suppressed in early pregnancy. Elevated levels of anandamide (AEA), the endogenous ligand for the CB1 and CB2 receptors (R), interfere with receptivity of the blastocyst. Ang-(1–7) is down-regulated in the implantation site (IS) in normal pregnancy at day 7 of gestation. We determined the effects of intra-uterine angiotensin-(1–7) [Ang-(1–7)] (24 microg/kg/h) or vehicle given into the left uterine horn on the ECs in the decidualized uterus.
Ovariectomized rats were sensitized for the decidual cell reaction by steroid treatment and decidualization was induced by a bolus of oil injected into the left horn; the right horn served as a control.
Decidualization increased endometrial permeability (3.1+/−0.2 vs. 7.1+/−0.5 uterus/muscle of cpm of (125)I-BSA, p < 0.0001). VEGF mRNA was increased by the decidualization (1.4-fold, p < 0.05) and by Ang-(1–7) (2.0-fold, p < 0.001). CB1R mRNA was reduced by decidualization (2.7-fold, p < 0.001), but increased by Ang-(1–7) (1.9-fold, p < 0.05). CB2R mRNA was increased by decidualization (4-fold, p < 0.05) and by Ang-(1–7) (2.4-fold, p < 0.001). The enzyme metabolizing AEA, fatty acid amide hydrolase (FAAH), was reduced by decidualization (7.8 fold, p < 0.001) and unchanged by Ang-(1–7) (p > 0.05), whereas the enzyme metabolizing 2-arachidonoylglycerol, monoacyl glycerol lipase (MAGL), was unchanged by decidualization (p > 0.05) and increased by Ang-(1–7) (1.7 fold, p < 0.001).
These findings report for the first time that Ang-(1–7) augments the expression of CB1R, CB2R and MAGL in the decidualized uterus and thus may interfere with the early events of decidualization.
PMCID: PMC4325957  PMID: 25596750
Pregnancy; Implantation; Decidualization; Anandamide; 2-arachidonoylglycerol; Angiotensin-(1–7); Pseudopregnancy
2.  Urinary phthalate metabolites in relation to maternal serum thyroid and sex hormone levels during pregnancy: a longitudinal analysis 
Increasing scientific evidence suggests that exposure to phthalates during pregnancy may be associated with an elevated risk of adverse reproductive outcomes such as preterm birth. Maternal endocrine disruption across pregnancy may be one pathway mediating some of these relationships. We investigated whether urinary phthalate metabolites were associated with maternal serum thyroid (free thyroxine [FT4], free triiodothyronine [FT3], and thyroid-stimulating hormone [TSH]), and sex (estradiol, progesterone, and sex hormone-binding globulin [SHBG]) hormone levels at multiple time points during pregnancy.
Preliminary data (n = 106) were obtained from an ongoing prospective birth cohort in Northern Puerto Rico. We collected urine and serum sample at the first and third study visits that occurred at 18 +/- 2 and 26 +/- 2 weeks of gestation, respectively. To explore the longitudinal relationships between urinary phthalate metabolites and serum thyroid and sex hormone concentrations, we used linear mixed models (LMMs) adjusted for prepregnancy body mass index (BMI) and maternal age. An interaction term was added to each LMM to test whether the effect of urinary phthalate metabolites on serum thyroid and sex hormone levels varied by study visit. In cross-sectional analyses, we stratified BMI- and age-adjusted linear regression models by study visit.
In adjusted LMMs, we observed significant inverse associations between mono-3-carboxypropyl phthalate (MCPP) and FT3 and between mono-ethyl phthalate (MEP) and progesterone. In cross-sectional analyses by study visit, we detected stronger and statistically significant inverse associations at the third study visit between FT3 and MCPP as well as mono-carboxyisooctyl phthalate (MCOP); also at the third study visit, significant inverse associations were observed between FT4 and metabolites of di-(2-ethylhexyl) phthalate (DEHP). The inverse association between MEP and progesterone was consistent across study visits.
In this group of pregnant women, urinary phthalate metabolites may be associated with altered maternal serum thyroid and sex hormone levels, and the magnitude of these effects may depend on the timing of exposure during gestation.
Electronic supplementary material
The online version of this article (doi:10.1186/1477-7827-13-4) contains supplementary material, which is available to authorized users.
PMCID: PMC4326411  PMID: 25596636
Biomarkers; Endocrine disruption; Epidemiology; Thyroid; Hormones; Urinary metabolites; Pregnancy; Phthalates
3.  Molecular cloning and expression analysis of dmrt1 and sox9 during gonad development and male reproductive cycle in the lambari fish, Astyanax altiparanae 
The dmrt1 and sox9 genes have a well conserved function related to testis formation in vertebrates, and the group of fish presents a great diversity of species and reproductive mechanisms. The lambari fish (Astyanax altiparanae) is an important Neotropical species, where studies on molecular level of sex determination and gonad maturation are scarce.
Here, we employed molecular cloning techniques to analyze the cDNA sequences of the dmrt1 and sox9 genes, and describe the expression pattern of those genes during development and the male reproductive cycle by qRT-PCR, and related to histology of the gonad.
Phylogenetic analyses of predicted amino acid sequences of dmrt1 and sox9 clustered A. altiparanae in the Ostariophysi group, which is consistent with the morphological phylogeny of this species. Studies of the gonad development revealed that ovary formation occurred at 58 days after hatching (dah), 2 weeks earlier than testis formation. Expression studies of sox9 and dmrt1 in different tissues of adult males and females and during development revealed specific expression in the testis, indicating that both genes also have a male-specific role in the adult. During the period of gonad sex differentiation, dmrt1 seems to have a more significant role than sox9. During the male reproductive cycle dmrt1 and sox9 are down-regulated after spermiation, indicating a role of these genes in spermatogenesis.
For the first time the dmrt1 and sox9 were cloned in a Characiformes species. We show that both genes have a conserved structure and expression, evidencing their role in sex determination, sex differentiation and the male reproductive cycle in A. altiparanae. These findings contribute to a better understanding of the molecular mechanisms of sex determination and differentiation in fish.
PMCID: PMC4298075  PMID: 25577427
Teleostei; Sex differentiation; DMRT1; SOX9; Spermatogenesis
4.  Is it time for a paradigm shift in understanding embryo selection? 
Embryo selection has been an integral feature of in vitro fertilization (IVF) almost since its inception. Since the advent of extended blastocyst stage embryo culture, and especially with increasing popularity of elective single embryo transfer (eSET), the concept of embryo selection has increasingly become a mainstay of routine IVF.
We here, however, argue that embryo selection via blastocyst stage embryo transfer (BSET), as currently practiced, at best improves IVF outcomes only for a small minority of patients undergoing IVF cycles. For a large majority BSET is either ineffective or, indeed, may actually be harmful by decreasing IVF pregnancy chances. Overall, only a small minority of patients, thus, benefit from prolonged embryo culture, while BSET, as a tool to enhance IVF outcomes, is increasingly utilized as routine care in IVF for all patients.
Since newer methods of embryo selection, like preimplantation genetic screening (PGS) and closed system embryo incubation with time-lapse photography are practically dependent on BSET, these concepts of embryo selection, currently increasingly adopted in mainstream IVF, require reconsideration. They, automatically, transfer the downsides of BSET, including decreases in IVF pregnancy chances in some patients, to these new procedures, and in addition raise serious questions about cost-effectiveness.
PMCID: PMC4326369  PMID: 25577140
5.  Cyclic guanosine monophosphate does not inhibit gonadotropin-induced activation of mitogen-activated protein kinase 3/1 in pig cumulus-oocyte complexes 
Recent results indicate a key role for cyclic guanosine monophosphate (cGMP) in the regulation of oocyte meiotic arrest in preovulatory mammalian follicles. The aim of our study was to determine whether the resumption of oocyte meiosis and expansion of cumulus cells in isolated pig cumulus-oocyte complexes (COCs) can be blocked by a high intracellular concentration of cGMP, and whether this effect is mediated by a cGMP-dependent inhibition of mitogen-activated protein kinase 3/1 (MAPK3/1).
The COCs were isolated from ovaries of slaughtered gilts and cultured in vitro in M199 supplemented with 5% fetal calf serum. The expression levels of the C-type natriuretic peptide (CNP) precursor (NPPC) and its receptor (NPR2) mRNAs during the culture of COCs were determined by real-time RT-PCR. To control the intracellular concentration of cGMP in the COCs, the culture medium was further supplemented with CNP or various concentrations of synthetic cGMP analogues; the concentration of cGMP in COCs was then assessed by ELISA. The effect of the drugs on oocyte maturation was assessed after 24 and 44 h of culture by determining nuclear maturation. The expansion of cumulus cells was assessed by light microscopy and the expression of cumulus expansion-related genes by real-time RT-PCR. A possible effect of cGMP on FSH-induced activation of MAPK3/1 was assessed by immunoblotting the COC proteins with phospho-specific and total anti-Erk1/2 antibodies.
The COCs expressed NPPC and NPR2, the key components of cGMP synthesis, and produced a large amount of cGMP upon stimulation with exogenous CNP, which lead to a significant (P < 0.05) delay in oocyte meiotic resumption. The COCs also responded to cGMP analogues by inhibiting the resumption of oocyte meiosis. The inhibitory effect of cGMP on meiotic resumption was reversed by stimulating the COCs with FSH. However, high concentration of intracellular cGMP was not able to suppress FSH-induced activation of MAPK3/1 in cumulus cells, cumulus expansion and expression of expansion-related genes (P > 0.05).
The findings of this study indicate that high cGMP concentrations inhibit the maturation of pig oocytes in vitro but the inhibitory mechanism does not involve the suppression of MAPK3/1 activation in cumulus cells.
PMCID: PMC4293816  PMID: 25567742
Pig; Oocyte; cGMP; Meiotic resumption; Expansion-related genes; MAPK3/1
6.  Assessment of ovarian reserve using anti-Müllerian hormone levels in benign gynecologic conditions and surgical interventions: a systematic narrative review 
The usefulness of anti-Müllerian hormone (AMH) for the quantitative evaluation of ovarian reserve has been established. Therefore, serum AMH has been recently applied to the assessment of ovarian reserve outside infertility treatment. We conducted a computer-based search, using keywords, through the PubMed database from inception until May 2014 and summarized available studies evaluating ovarian damage caused by gynecologic diseases, such as endometriosis and ovarian tumor, as well as surgical interventions, such as cystectomy and uterine artery embolization (UAE), to discuss the usefulness of serum AMH. Most of the studies demonstrated a decline of serum AMH levels after cystectomy for endometriomas. It is not conclusive whether electrocoagulation or suturing is preferable. The effects of other gynecologic diseases and interventions, such as hysterectomy and UAE, on ovarian reserve are controversial. Serum AMH levels should be considered in determining the indication and selection of operative methods for benign gynecologic conditions.
PMCID: PMC4274680  PMID: 25510324
AMH; Cystectomy; Endometriosis; Ovarian reserve; UAE
7.  Establishment of comparative performance criteria for IVF centers: correlation of live birth rates in autologous and donor oocyte IVF cycles 
To assess whether an objective performance criterion for in vitro fertilization (IVF) centers can be established.
A retrospective analysis of 2011 National ART Surveillance System data for 451 U.S. IVF centers, 137 of which were included in the analysis since they performed >20 fresh embryo transfers per age group and >20 fresh oocyte donor transfers. The analysis of autologous cycles was restricted to women under age 40. The main outcome measure was correlation between center-specific live birth rates (LBR) in autologous and donor oocyte cycles.
55.6% donor and 46.7%, 39.1% and 28.7% (for ages <35, 35–37 and 38–40 years) autologous cycles resulted in live births per fresh embryo transfer. Donor LBR predicted autologous LBR (< 35 years, P < 0.001; 35 – 38 years, P < 0.001; 38 – 40 years, P = 0.015). Clinics with high prevalence of patients with diminished ovarian reserve had lower autologous LBR per age group (P = 0.015). Every 10% increase in donor LBR increased odds of autologous LBR above the age-adjusted national average by 68% (OR 1.68; 95% CI 1.36 – 2.07; P < 0.001).
Since center-specific donor and autologous IVF cycle outcomes correlate, and as donor cycles reflect fewer patient covariates, they represent a first comparable performance measure between centers, allowing for internal as well as external quality control.
PMCID: PMC4267438  PMID: 25475407
Assisted reproductive technology (ART); In vitro fertilization (IVF); Live birth rates; Donor oocyte cycles; Quality controls
8.  IGFBP-4 and −5 are expressed in first-trimester villi and differentially regulate the migration of HTR-8/SVneo cells 
Adverse gestational outcomes such as preeclampsia (PE) and intrauterine growth restriction (IUGR) are associated with placental insufficiency. Normal placental development relies on the insulin-like growth factors -I and -II (IGF-I and -II), in part to stimulate trophoblast proliferation and extravillous trophoblast (EVT) migration. The insulin-like growth factor binding proteins (IGFBPs) modulate the bioavailability of IGFs in various ways, including sequestration, potentiation, and/or increase in half-life. The roles of IGFBP-4 and −5 in the placenta are unknown, despite consistent associations between pregnancy complications and the levels of two IGFBP-4 and/or −5 proteases, pregnancy-associated plasma protein -A and -A2 (PAPP-A and PAPP-A2). The primary objective of this study was to elucidate the effects of IGFBP-4 and −5 on IGF-I and IGF-II in a model of EVT migration. A related objective was to determine the timing and location of IGFBP-4 and −5 expression in the placental villi.
We used wound healing assays to examine the effects of IGFBP-4 and −5 on the migration of HTR-8/SVneo cells following 4 hours of serum starvation and 24 hours of treatment. Localization of IGFBP-4, −5 and PAPP-A2 was assessed by immunohistochemical staining of first trimester placental sections.
2 nM IGF-I and -II each increased HTR-8/SVneo cell migration with IGF-I increasing migration significantly more than IGF-II. IGFBP-4 and −5 showed different levels of inhibition against IGF-I. 20 nM IGFBP-4 completely blocked the effects of 2 nM IGF-I, while 20 nM IGFBP-5 significantly reduced the effects of 2 nM IGF-I, but not to control levels. Either 20 nM IGFBP-4 or 20 nM IGFBP-5 completely blocked the effects of 2 nM IGF-II. Immunohistochemistry revealed co-localization of IGFBP-4, IGFBP-5 and PAPP-A2 in the syncytiotrophoblast layer of first trimester placental villi as early as 5 weeks of gestational age.
IGFBP-4 and −5 show different levels of inhibition on the migration-stimulating effects of IGF-I and IGF-II, suggesting different roles for PAPP-A and PAPP-A2. Moreover, co-localization of the pappalysins and their substrates within placental villi suggests undescribed roles of these molecules in early placental development.
Electronic supplementary material
The online version of this article (doi:10.1186/1477-7827-12-123) contains supplementary material, which is available to authorized users.
PMCID: PMC4271501  PMID: 25475528
Pappalysins; PAPP-A; PAPP-A2; Insulin-like growth factor-binding proteins; IGFBP-4; IGFBP-5; Trophoblast migration
9.  Sperm quality after density gradient centrifugation with three commercially available media: a controlled trial 
Density gradient is the preferred technique for sperm processing for ART. However, no study has examined sperm quality using different processing media simultaneously and under identical conditions. Therefore, we evaluated semen quality following sperm preparation by three commonly used commercially available density gradient media in a well-designed controlled trial.
We obtained semen samples from 20 healthy volunteers. Percent motility, total motile sperm (TMS), % recovery and DNA damage were assessed before and after separation in three different sperm density gradient media-PureCeption, ISolate and SpermGrad-125.
Percent motility was higher in the ISolate (81.4% ± 6.6%) and SpermGrad-125 samples (85.7% ± 8.0%) (P < 0.0001) than in the PureCeption samples (62.5% ± 13.2%) (P = 0.07). TMS was higher in the PureCeption(TM) and ISolate samples (14.2% ± 15.9% and 15.8% ± 18.2%) than in those prepared with SpermGrad-125 (10.6% ± 19.7%) (P < 0.0001). Percent recovery was significantly higher in the PureCeption(TM) and ISolate samples (45.3% and 48.9%) than in the SpermGrad-125(TM) samples (30.8%) (P < 0.01). DNA fragmentation was comparable across the three gradients (PureCeption = 8.8% ± 4.7%; ISolate = 7.2 ± 5.2% and SpermGrad-125 = 11.2% ± 7.4%).
Three different density gradient processing media PureCeption, ISolate, and SpermGrad-125 were examined for their effects on sperm quality. Sperm processed by ISolate and Sperm Grad 125 had better motility and TMS after processing. The extent of DNA damage was comparable in all three gradients.
PMCID: PMC4271431  PMID: 25466430
Sperm motility; Sperm processing; Reproductive potential; Density gradient centrifugation; DNA fragmentation
10.  Ovarian reserve markers and assisted reproductive technique (ART) outcomes in women with advanced endometriosis 
The role of ovarian reserve markers as predictors of the controlled ovarian stimulation (COS) response in intracytoplasmic sperm injection (ICSI) cycles in women with endometriosis has been much debated. The aim of the present study is to assess the predictability of ovarian reserve markers for the number of mature oocytes (MII) retrieved and to assess the pregnancy rate and live birth rate in women with advanced endometriosis.
Two hundred eighty-five infertile women who had laparoscopy followed by a first ICSI cycle were recruited in this prospective study. One hundred ten patients were diagnosed with endometriosis stage III-IV (group 1), and 175 patients had no endometriosis (group II). Sixty-three patients in group 1 had no history of previous endometrioma surgery (group Ia), and 47 patients had a history of previous endometrioma surgery (group Ib).
The number of mature oocytes retrieved was significantly lower in women with advanced endometriosis than in women with no endometriosis. The number of mature oocytes retrieved in women with and without endometriosis was best predicted by antral follicle count (AFC) and age, whereas only AFC was a predictor in women with previous endometrioma surgery (odds ratio: 0.49; 95% confidence interval: 0.13-0.60). Women with endometriosis had a lower rate of live births than the control group, but this difference was not statistically significant; the number of live births was significantly lower in those with previous endometrioma surgery.
The best predictor of the COS response in ICSI was AFC, followed by age. Women receiving ICSI following surgery for ovarian endometrioma had a poorer clinical outcome and lower rate of live births compared with those with endometriosis but no previous surgery and the control group.
PMCID: PMC4266956  PMID: 25442239
Stage III-IV endometriosis; Antral follicle count (AFC); Mature oocyte; Intracytoplasmic sperm injection (ICSI); Live birth rate
11.  In high responding patients undergoing an initial IVF cycle, elevated estradiol on the day of hCG has no effect on live birth rate 
The impact of elevated estradiol on the day of human chorionic gonadotropin (hCG) administration on in vitro fertilization (IVF) outcomes has been debated for over 25 years. Some investigators have shown a positive effect, others a negative effect; while most have shown no effect. Few studies have expressed their findings based on live birth. This study examined the relationship between estradiol level and other IVF cycle response parameters in relation to pregnancy, with a focus on live births after controlling for embryo quality.
We performed a retrospective cohort study on 489 patients <40 years old that underwent an initial IVF cycle. Estradiol concentration on the day of hCG was categorized as; low <2000 pg/ml), mid (2001-4000 pg/ml) and high (>4000 pg/ml) to determine how estradiol level on the day of hCG affected response variables during the IVF cycle. We performed a subgroup analysis restricted to patients with good/fair quality embryos transferred (n = 428), to control for embryo quality and assessed pregnancy outcome. The association between estradiol and live birth (LB) was then evaluated after identifying and controlling for confounding factors. Multivariate analysis was used to identify significant main effects and interactions in the model. Estradiol levels were also compared in patients having a LB or not (NLB) in both populations.
We found that estradiol was significantly related to + hCG, clinical pregnancy rate, age, and most other IVF cycle response variables. After performing the subgroup analysis controlling for embryo quality, we found that LB rates were not different. Only the main effects of average embryo quality at transfer (AEQS), age and transferring two embryos influenced LB. Estradiol levels were also compared in patients having a LB or NLB in both populations and was found to be higher/not different in LB patients. LB rates and AEQS were also not different in a subgroup of patients having an elevated level of estradiol (>4200 pg/ml) on the day of hCG in patients having embryo transfer on day 3 or day 5.
After controlling for embryo quality, elevated estradiol on the day of hCG had no effect on LB.
PMCID: PMC4258294  PMID: 25430541
IVF; Estradiol level; High responder; Live birth rate; Age; Embryo quality; Day of embryo transfer
12.  Suitable housekeeping genes for normalization of transcript abundance analysis by real-time RT-PCR in cultured bovine granulosa cells during hypoxia and differential cell plating density 
Bovine granulosa cell culture models are important to understand molecular mechanisms of ovarian function. Folliculogenesis and luteinization are associated with increasing density of cells and local hypoxic conditions. The current study identified two reliable housekeeping genes useful for gene normalization in granulosa cells under different in vitro conditions.
During the current experiments cells were subjected to different biological and physical stimuli, follicle stimulating hormone, different initial cell plating density and hypoxia. Transcript abundance of seven housekeeping genes was quantified by real-time RT-PCR with co-amplification of the respective external standard.
Three of the genes, GAPDH, HMBS, and HPRT1 were found to be regulated by initial cell plating density, five of them, GAPDH, HMBS, HPRT1, RPLP0 and RPS18 under hypoxic conditions, but none of them after FSH stimulation. In detail, GAPDH was up regulated, but HPRT1 and HMBS were down regulated at high density and under hypoxia. Expression of RPLP0 and RPS18 was inconsistent, but was significantly down-regulated in particular at high cell density combined with hypoxia. In contrast, TBP and B2M genes were neither regulated under different plating density conditions nor by hypoxia as they showed similar expression levels under all conditions analyzed.
The present data indicate that TBP and B2M are appropriate housekeeping genes for normalization of transcript abundance measured by real-time RT-PCR in granulosa cells subjected to different plating densities, oxygen concentrations and FSH stimulation.
PMCID: PMC4280684  PMID: 25430436
TBP; B2M; GAPDH; HMBS; RPS18; RPLP0; HPRT1; External standards; Co-amplification
13.  Usefulness of bovine and porcine IVM/IVF models for reproductive toxicology 
Women presenting fertility problems are often helped by Assisted Reproductive Techniques (ART), such as in vitro fertilization (IVF) programs. However, in many cases the etiology of the in/subfertility remains unknown even after treatment. Although several aspects should be considered when assisting a woman with problems to conceive, a survey on the patients’ exposure to contaminants would help to understand the cause of the fertility problem, as well as to follow the patient properly during IVF. Daily exposure to toxic compounds, mainly environmental and dietary ones, may result in reproductive impairment. For instance, because affects oocyte developmental competence. Many of these compounds, natural or synthetic, are endocrine disruptors or endocrine active substances that may impair reproduction. To understand the risks and the mechanism of action of such chemicals in human cells, the use of proper in vitro models is essential. The present review proposes the bovine and porcine models to evaluate toxic compounds on oocyte maturation, fertilization and embryo production in vitro. Moreover, we discuss here the species-specific differences when mice, bovine and porcine are used as models for human.
PMCID: PMC4258035  PMID: 25427762
Female fertility; Toxicology; Bovine; Porcine; Model
14.  Role of activins in embryo implantation and diagnosis of ectopic pregnancy: a review 
Embryo implantation is a major prerequisite for the successful establishment of pregnancy. Ectopic implantation outside the intrauterine cavity and the development of ectopic pregnancy (EP) is a major cause of maternal morbidity and occasionally mortality during the first trimester. EP may be induced by failure of tubal transport and/or increased tubal receptivity. Activins, their type II receptors and follistatin have been localised in the human endometrial and tubal epithelium and they are major regulators of endometrial and tubal physiology during the menstrual cycle. Pathological expression of activins and their binding protein, follistatin, was observed in tissue and serum samples collected from EP. Several studies with different designs investigated the diagnostic value of a single measurement of serum activin-A in the differentiation between normal intrauterine and failing early pregnancy and the results are controversial. Nevertheless, the diagnostic value of activins in EP, including the other activin isoforms (activin-B and –AB) and follistatin, merits further research. This review appraises the data to date researching the role of activins in the establishment of normal pregnancy and, pathogenesis and diagnosis of tubal EP.
PMCID: PMC4254208  PMID: 25421645
Activin; Fallopian tube; Endometrium; Implantation; Ectopic pregnancy; Early diagnosis
15.  The presence of multiple gestational sacs confers a higher live birth rate in women with infertility who achieve a positive pregnancy test after fresh and frozen embryo transfer: a retrospective local cohort 
After spontaneous conception, the rate of miscarriage is more common in multiple rather than singleton pregnancies. However, the incidence of miscarriage is lower in in-vitro fertilization twin versus singleton pregnancies. Most patients have little understanding of pregnancy outcomes once they achieve a positive pregnancy test. This study investigated the relationship between multiple pregnancy and miscarriage in women with infertility after fresh and frozen embryo transfer.
Retrospective local cohort study of all consecutive patients undergoing in-vitro fertilization at our institution (n = 1130), fresh or frozen embryo transfer, between January 1, 2008 and December 31, 2012. Patient characteristics (age, body mass index, initial hCG, maximum follicle stimulating hormone levels) and in-vitro fertilization parameters (estradiol levels, eggs retrieved, and endometrial thickness) were collected and statistically analyzed using T-test and Chi-square test (Stata version 10). Linear and logistic regression were used when appropriate.
Overall, live birth rate for all cycles was 30.44% and total pregnancy loss was 6.55% - similar for fresh and frozen cycles despite a higher rate of biochemical pregnancies for frozen cycles. Among all pregnant patients, 62.48% had a live birth. Although clinical pregnancy rate was higher for fresh cycles, live birth rates were similar. In pregnancies where multiple sacs were demonstrated on ultrasound, live birth rates were higher despite 31% of patients losing at least one sac. This finding was comparable between fresh and frozen cycles. However, in patients under age 35 and using donor egg, no live birth advantage was seen in patients with multiple sacs. In fact, transferring more than one embryo did not increase live birth rate either.
Despite the many maternal and fetal risks of multiple pregnancies, patients who achieve a positive pregnancy test with fresh and frozen in-vitro fertilization and who have more than one pregnancy sac are more likely ultimately to deliver at least one baby. This finding is true of both fresh and frozen embryo transfer cycles. This pregnancy advantage is not seen in young patients and in patients using donor egg, and single embryo transfer maximizes birth outcomes.
PMCID: PMC4247647  PMID: 25422171
In-vitro fertilization (IVF); Frozen embryo transfer (FET); Miscarriage; Multiple pregnancy
16.  In vitro maturation is slowed in prepubertal lamb oocytes: ultrastructural evidences 
In vitro maturation (IVM) of immature oocytes retrieved from unstimulated ovaries may avoid side effects connected to hyperstimulation during IVF procedures, including the risk of cancer recurrence. In humans, the scarce availability of immature oocytes limits morphological studies. The monovular ovine may represent an experimental model for IVM studies.
To assess if the scarce developmental competence of prepubertal oocytes (PO) is related to morphological changes we analyzed, by light and transmission electron microscopy, cumulus-oocyte-complexes (COCs) from lambs (30-40 days old) and sheep (4-6 years old) at sampling and after 7 h, 19 h, 24 h of IVM. Meiotic progression was determined at the same time points.
At sampling, the germinal vesicle (GV) of PO was round and centrally or slightly eccentrically located, whereas in adult oocytes (AO) it was irregularly shaped and flattened against the oolemma. PO, differently from AO, showed numerous trans-zonal projections. Organelles, including cortical granules (CGs), were more abundant in AO. After 7 h, the percentage of AO that underwent GVBD-MI transition increased significantly. In PO, the oolemma was juxtaposed to the ZP; in AO, it showed several spikes in correspondence of cumulus cells (CC) endings. In PO, organelles and isolated CGs were scattered in the ooplasm. In AO, groups of CGs were also present under the oolemma. After 19 h, PO underwent GVBD-MI transition; their oolemma showed several spikes, with CC projections retracted and detached from the ZP. AO underwent MI-MII transition; their oolemma regained a round shape. CGs were located beneath the plasmalemma, arranged in multiple, continuous layers, sometime discontinuous in PO. After 24 h, both groups reached the MII-stage, characterized by a regular oolemma and by expanded CCs. PO showed CGs distributed discontinuously beneath the oolemma, while AO showed a continuous monolayer of CGs.
Even if PO were able of reaching morphological maturation after 24 h of IVM, our ultrastructural analysis allowed detecting the presumptive sequence of cytoplasmic alterations connected with the delay of nuclear maturation, that might explain the reduced developmental competence of such oocytes. Data from the sheep model are of interest for zootechny, and provide an experimental basis for improving human IVM technology.
PMCID: PMC4247763  PMID: 25421574
Immature oocyte; Cumulus-oocyte complexes; In vitro maturation; Ultrastructure; Lamb; Sheep
17.  International regulatory landscape and integration of corrective genome editing into in vitro fertilization 
Genome editing technology, including zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeat (CRISPR)/Cas, has enabled far more efficient genetic engineering even in non-human primates. This biotechnology is more likely to develop into medicine for preventing a genetic disease if corrective genome editing is integrated into assisted reproductive technology, represented by in vitro fertilization. Although rapid advances in genome editing are expected to make germline gene correction feasible in a clinical setting, there are many issues that still need to be addressed before this could occur. We herein examine current status of genome editing in mammalian embryonic stem cells and zygotes and discuss potential issues in the international regulatory landscape regarding human germline gene modification. Moreover, we address some ethical and social issues that would be raised when each country considers whether genome editing-mediated germline gene correction for preventive medicine should be permitted.
Electronic supplementary material
The online version of this article (doi:10.1186/1477-7827-12-108) contains supplementary material, which is available to authorized users.
PMCID: PMC4251934  PMID: 25420886
Genome editing; ZFN; TALEN; CRISPR/Cas; Embryonic stem cells; Zygote; Embryo; Assisted reproductive technology; In vitro fertilization; Genetic disease; Prevention; Germline gene modification; Regulations
18.  Recombinant luteinizing hormone supplementation in women undergoing in vitro fertilization/ intracytoplasmic sperm injection with gonadotropin releasing hormone antagonist protocol: a systematic review and meta-analysis 
The objective of this meta-analysis is to assess the impact of LH supplementation in women undergoing in vitro fertilization/ intracytoplasmic sperm injection (IVF/ICSI) with gonadotropin releasing hormone (GnRH) antagonist protocol. No significant difference in outcomes between LH supplementation and r-FSH alone in women undergoing IVF/ICSI with GnRH antagonist protocol is currently present, and further studies are necessary for more solid conclusions on pregnancy likelihood to be drawn.
PMCID: PMC4255634  PMID: 25420965
Recombinant FSH; Recombinant LH; Ovarian stimulation; Meta-analysis; Oral contraceptive pills
19.  Making gametes from pluripotent stem cells – a promising role for very small embryonic-like stem cells 
The urge to have one’s own biological child supersedes any desire in life. Several options have been used to obtain gametes including pluripotent stem cells (embryonic ES and induced pluripotent iPS stem cells); gonadal stem cells (spermatogonial SSCs, ovarian OSCs stem cells), bone marrow, mesenchymal cells and fetal skin. However, the field poses a huge challenge including inefficient existing protocols for differentiation, epigenetic and genetic changes associated with extensive in vitro manipulation and also ethical/regulatory constraints. A tremendous leap in the field occurred using mouse ES and iPS cells wherein they were first differentiated into epiblast-like cells and then primordial germ cell-like cells. These on further development produced sperm, oocytes and live offspring (had associated genetic problems). Evidently differentiating pluripotent stem cells into primordial germ cells (PGCs) remains a major bottleneck. Against this backdrop, we propose that a novel population of pluripotent stem cells termed very small embryonic-like stem cells (VSELs) may serve as an alternative, potential source of autologus gametes, keeping in mind that they are indeed PGCs surviving in adult mammalian ovaries and testes. Both VSELs and PGCs are pluripotent, relatively quiescent because of epigenetic modifications of parentally imprinted genes loci like Igf2-H19 and KCNQ1p57, share several markers like Stella, Fragilis, Mvh, Dppa2, Dppa4, Sall4, Blimp1 and functional receptors. VSELs are localized in the basement membrane of seminiferous tubules in testis and in the ovary surface epithelium. Ovarian stem cells from mouse, rabbit, sheep, marmoset and humans (menopausal women and those with premature ovarian failure) spontaneously differentiate into oocyte-like structures in vitro with no additional requirement of growth factors. Thus a more pragmatic option to obtain autologus gametes may be the pluripotent VSELs and if we could manipulate them in vivo – existing ethical and epigenetic/genetic concerns associated with in vitro culture may also be minimized. The field of oncofertility may undergo a sea-change and existing strategies of cryopreservation of gametes and gonadal tissue for fertility preservation in cancer patients will necessitate a revision. However, first the scientific community needs to arrive at a consensus about VSELs in the gonads and then work towards exploiting their potential.
PMCID: PMC4255929  PMID: 25421462
PGCs; Germ cells; Gametes; Sperm; Oocyte; ES cells; iPS cells; VSELs; Infertility; Assisted reproduction
20.  Fine morphological assessment of quality of human mature oocytes after slow freezing or vitrification with a closed device: a comparative analysis 
Human mature oocytes are very susceptible to cryodamage. Several reports demonstrated that vitrification might preserve oocyte better than slow freezing. However, this is still controversial. Thus, larger clinical, biological and experimental trials to confirm this concept are necessary. The aim of the study was to evaluate and compare fine morphological features in human mature oocytes cryopreserved with either slow freezing or vitrification.
We used 47 supernumerary human mature (metaphase II) oocytes donated by consenting patients, aged 27-32 years, enrolled in an IVF program. Thirtyfive oocytes were cryopreserved using slow freezing with 1.5 M propanediol +0.2 M sucrose concentration (20 oocytes) or a closed vitrification system (CryoTip Irvine Scientific CA) (15 oocytes). Twelve fresh oocytes were used as controls. All samples were prepared for light and transmission electron microscopy evaluation.
Control, slow frozen/thawed and vitrified/warmed oocytes (CO, SFO and VO, respectively) were rounded, 90–100 μm in diameter, with normal ooplasm showing uniform distribution of organelles. Mitochondria-smooth endoplasmic reticulum (M-SER) aggregates and small mitochondria-vesicle (MV) complexes were the most numerous structures found in all CO, SFO and VO cultured for 3–4 hours. M-SER aggregates decreased, and large MV complexes increased in those SFO and VO maintained in culture for a prolonged period of time (8–9 hours). A slight to moderate vacuolization was present in the cytoplasm of SFO. Only a slight vacuolization was present in VO, whereas vacuoles were almost completely absent in CO. Amount and density of cortical granules (CG) appeared abnormally reduced in SFO and VO, irrespective of the protocol applied.
Even though, both slow freezing and vitrification ensured a good overall preservation of the oocyte, we found that: 1) prolonged culture activates an intracellular membrane “recycling” that causes the abnormal transformation of the membranes of the small MV complexes and of SER into larger rounded vesicles; 2) vacuolization appears as a recurrent form of cell damage during slow freezing and, at a lesser extent, during vitrification using a closed device; 3) premature CG exocytosis was present in both SFO and VO and may cause zona pellucida hardening.
PMCID: PMC4255960  PMID: 25421073
Oocyte; Cryopreservation; Slow freezing; Vitrification; Ultrastructure; Human
21.  A critical review of bi-dimensional and three-dimensional ultrasound techniques to monitor follicle growth: do they help improving IVF outcome? 
This review focuses on the possibility of improving the outcome of human IVF by studying the follicles where oocytes grow by ultrasound techniques. A comprehensive analysis of bi-dimensional (2D) and three-dimensional (3D) ultrasound (US) assessment of the follicle size and volume is presented.
Published reports from the year 1999 to 2014 analyzing the relationship between oocyte competence, IVF outcome and ultrasound assessment of the follicle size and volume have been critically analyzed.
US assessment of growing follicles has been performed mainly by 2D-US, and while overall very useful, it has been found to be of limited usefulness in predicting oocyte competence, recognize which follicles will release a mature metaphase II oocytes and decide the ideal time to trigger ovulation. In fact, a quite wide follicle size range (16–22 mm) has been reported to be associated with mature oocytes with good competence toward fertilization and embryo development. It has been also shown that smaller follicles sometimes contain mature, fertilizable oocytes. However, embryos derived from smaller follicles have probably a lower implantation potential, while follicles larger than 22 mm often contain post-mature eggs.
The study of follicular size by 2D-US is of limited usefulness in helping in the identification of follicles containing the best oocytes and in choosing the best moment to trigger ovulation. Possibly the value of US in this area will be improved by large prospective studies in which automated 3D-US will be used.
PMCID: PMC4255967  PMID: 25420733
Follicle size; Follicle volume; Oocyte competence; 3D ultrasound; SonoAVC; IVF outcome
22.  The effectiveness of flow cytometric sorting of human sperm (MicroSort®) for influencing a child’s sex 
Flow cytometric sorting can be used to separate sperm based on sex chromosome content. Differential fluorescence emitted by stained X- vs. Y-chromosome-bearing sperm enables sorting and collection of samples enriched in either X- or Y-bearing sperm for use to influence the likelihood that the offspring will be a particular sex. Herein we report the effectiveness of flow cytometric sorting of human sperm and its use in human ART procedures.
This prospective, observational cohort study of the series of subjects treated with flow cytometrically sorted human sperm was conducted at investigational sites at two private reproductive centers. After meeting inclusion criteria, married couples (n = 4993) enrolled to reduce the likelihood of sex-linked or sex-limited disease in future children (n = 383) or to balance the sex ratio of their children (n = 4610). Fresh or frozen-thawed semen was processed and recovered sperm were stained with Hoechst 33342 and sorted by flow cytometry (n = 7718) to increase the percentage of X-bearing sperm (n = 5635) or Y-bearing sperm (n = 2083) in the sorted specimen. Sorted sperm were used for IUI (n = 4448) and IVF/ICSI (n = 2957). Measures of effectiveness were the percentage of X- and Y-bearing sperm in sorted samples, determined by fluorescence in situ hybridization, sex of babies born, IVF/ICSI fertilization- and cleavage rates, and IUI, IVF/ICSI, FET pregnancy rates and miscarriage rates.
Sorted specimens averaged 87.7 ± 5.0% X-bearing sperm after sorting for X and 74.3 ± 7.0% Y-bearing sperm after sorting for Y. Seventy-three percent of sorts were for girls. For babies born, 93.5% were females and 85.3% were males after sorting for X- and Y-bearing sperm, respectively. IUI, IVF/ICSI, and FET clinical pregnancy rates were 14.7%, 30.8%, and 32.1%, respectively; clinical miscarriage rates were 15.5%, 10.2%, and 12.7%.
Flow cytometric sorting of human sperm shifted the X:Y sperm ratio. IUI, IVF/ICSI and FET outcomes were consistent with unimpaired sperm function. Results provide evidence supporting the effectiveness of flow cytometric sorting of human sperm for use as a preconception method of influencing a baby’s sex.
Trial registration
NCT00865735 (
PMCID: PMC4256056  PMID: 25420620
Human sperm, sperm sorting; Flow cytometry; Sex selection; IUI; IVF/ICSI; FET; ART procedures
23.  Array comparative genomic hybridization analyses of all blastomeres of a cohort of embryos from young IVF patients revealed significant contribution of mitotic errors to embryo mosaicism at the cleavage stage 
Embryos produced by in vitro fertilization (IVF) have a high level of aneuploidy, which is believed to be a major factor affecting the success of human assisted reproduction treatment. The aneuploidy rate of cleavage stage embryos based on 1–2 biopsied blastomeres has been well-reported, however, the true aneuploidy rate of whole embryos remain unclear because of embryo mosaicism. To study the prevalence of mosaicism in top quality IVF embryos, surplus embryos donated from young patients (aged 28–32) in the assisted reproduction program at Queen Mary Hospital, Hong Kong were used.
Thirty-six good quality day 2 embryos were thawed. Out of the 135 blastomeres in these embryos, 121 (89.6%) survived thawing. Twelve of these embryos without lysed blastomeres and which cleaved to at least seven cells after a 24-h culture were dissembled into individual blastomeres, which were analysed by array comparative genomic hybridization and microsatellite marker analysis by fluorescent PCR.
Out of 12 day-3 embryos, 2 (16.7%) were normal, 3 (25%) were diploid/aneuploidy with <38% abnormality, 4 (33.3%) were diploid/aneuploidy mosaic with > =38% abnormality, and three (25%) were mosaic aneuploids. Conclusive chromosomal data were obtained from a high percentage of blastomeres (92.8%, 90/97). Microsatellite marker analysis performed on blastomeres in aneuploid embryos enabled us to reconstruct the chromosomal status of the blastomeres in each cleavage division. The results showed the occurrence of meiotic errors in 3 (25%) of the studied embryos. There were 16 mitotic errors (18.8%, 16/85) in the 85 mitotic divisions undertaken by the studied embryos. The observed mitotic errors were mainly contributed by endoreduplication (31.3%, 5/16), non-disjunction (25%, 4/16) and anaphase lagging (25%, 4/16). Chromosome breakages occurred in 6 divisions (7.1%, 6/85).
Mosaicism occurs in a high percentage of good-quality cleavage stage embryos and mitotic errors contribute significantly to the abnormality.
Electronic supplementary material
The online version of this article (doi:10.1186/1477-7827-12-105) contains supplementary material, which is available to authorized users.
PMCID: PMC4256731  PMID: 25420429
Cleavage stage embryo; Mosaicism; Aneuploidy
24.  Utility of antioxidants during assisted reproductive techniques: an evidence based review 
Assisted reproductive technology (ART) is a common treatment of choice for many couples facing infertility issues, be it due to male or female factor, or idiopathic. Employment of ART techniques, however, come with its own challenges as the in vitro environment is not nearly as ideal as the in vivo environment, where reactive oxygen species (ROS) build-up leading to oxidative stress is kept in check by the endogenous antioxidants system. While physiological amounts of ROS are necessary for normal reproductive function in vivo, in vitro manipulation of gametes and embryos exposes these cells to excessive ROS production either by endogenous or exogenous environmental factors. In this review, we discuss the sources of ROS in an in vitro clinical setting and the influence of oxidative stress on gamete/embryo quality and the outcome of IVF/ICSI. Sources of ROS and different strategies of overcoming the excessive generation of ROS in vitro are also highlighted. Endogenously, the gametes and the developing embryo become sources of ROS. Multiple exogenous factors act as potential sources of ROS, including exposure to visible light, composition of culture media, pH and temperature, oxygen concentration, centrifugation during spermatozoa preparation, ART technique involving handling of gamete/embryo and cryopreservation technique (freeze/thawing process). Finally, the use of antioxidants as agents to minimize ROS generation in the in vitro environment and as oral therapy is highlighted. Both enzymatic and non-enzymatic antioxidants are discussed and the outcome of studies using these antioxidants as oral therapy in the male or female or its use in vitro in media is presented. While results of studies using certain antioxidant agents are promising, the current body of evidence as a whole suggests the need for further well-designed and larger scale randomized controlled studies, as well as research to minimize oxidative stress conditions in the clinical ART setting.
PMCID: PMC4258799  PMID: 25421286
Reactive oxygen species; Oxidative stress; Antioxidants; Assisted reproductive technology; In vitro fertilization; Intracytoplasmic sperm injection; ART outcome
25.  Differential expression of pluripotent and germ cell markers in ovarian surface epithelium according to age in female mice 
Many studies have proposed that putative ovarian stem cells (OSCs) derived from the ovarian surface epithelium (OSE) layer of adult mammalian ovaries can produce oocytes. Few studies have reported that ovaries of aged mammalian females including mice and women possess rare premeiotic germ cells that can generate oocytes. However, no studies have reported the changes of OSCs according to the age of the female. Therefore, this study evaluated pluripotent and germ cell marker expression in the intact ovary, scraped OSE, and postcultured OSE according to age in female mice.
C57BL/6 female mice of 2 age groups (6–8 and 28–31 weeks) were superovulated by injection with 5 IU equine chorionic gonadotropin (eCG). Both ovaries were removed after 48 hours and scrapped to obtain OSE. Gene expressions of pluripotent (Oct-4, Sox-2, Nanog) and germ cell markers (c-Kit, GDF-9, and VASA) were evaluated by RT-PCR. VASA and GDF-9 were immune-localized in oocyte-like structures.
Expressions of germ cell markers in the intact ovary were significantly decreased in aged females, whereas expressions of pluripotent markers were not detected, regardless of age. Scraped OSE expression of all pluripotent and germ cell markers, except for c-Kit, was similar between both age groups. Three weeks postcultured OSE had significantly decreased expression of GDF-9 and VASA , but not c-Kit, in old mice, as compared to young mice; however there was no difference in the expression of other genes. The number of positively stained Oct-4 by immunohistochemistry in postcultured OSE was 2.5 times higher in young mice than aged mice. Oocyte-like structure was spontaneously produced in postcultured OSE. However, while that of young mice revealed a prominent nucleus, zona pellucida-like structure and cytoplasmic organelles, these features were not observed in old mice.
These results show that aged female mice have putative OSCs in OSE, but their differentiation potential, as well as the number of OSCs differs from those of young mice.
PMCID: PMC4280751  PMID: 25421381
Putative ovarian stem cells (OSCs); Ovarian surface epithelium (OSE); Pluripotent and germ cell markers; Female age

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