Anti-Müllerian hormone and inhibin B as predictors of pregnancy after treatment by in vitro fertilization/intracytoplasmic sperm injection

OBJECTIVE: To evaluate anti-Müllerian hormone (AMH) as a marker of reproductive outcome after IVF/intracytoplasmic sperm injection (ICSI). DESIGN: Longitudinal study. SETTING: University hospital. PATIENT(S): Two hundred seventy-six consecutive women undergoing IVF/ICSI. INTERVENTION(S): Ovarian stimulation, oocyte retrieval, IVF, ICSI, embryo transfer, AMH, and inhibin B determinations in serum and follicular fluid (FF). MAIN OUTCOME MEASURE(S): The AMH and inhibin B concentrations in 276 matched FF/serum pairs have been determined. Different outcome groups have been compared and set in relation to the oocyte count, morphological parameters, and steroid hormone levels. RESULT(S): The concentrations of AMH and inhibin B in both serum and FF were significantly higher in the group of women who became pregnant in the corresponding treatment cycle than in those who did not conceive. Positive correlations were observed between serum inhibin B concentrations and embryo morphology (r = 0.126, 95% confidence interval 0.026-0.284). Serum and FF AMH or inhibin B correlated positively with the oocyte count and negatively with the pretreatment cycle day 3 FSH level and the total administered gonadotropin dose. CONCLUSION(S): The AMH and inhibin B levels on the day of oocyte retrieval are correlated to reproductive outcome.

Intravaginal and intracervical application of seminal plasma in in vitro fertilization or intracytoplasmic sperm injection treatment cycles--a double-blind, placebo-controlled, randomized pilot study

OBJECTIVE: To evaluate whether intravaginal application of seminal plasma at the time of follicle aspiration in IVF or intracytoplasmic sperm injection treatment cycles has the potential to increase pregnancy rate. To calculate the number of patients needed to achieve significance in a multicenter trial. DESIGN: Double-blind, placebo-controlled randomized pilot study. SETTING: University department of gynecological endocrinology and reproductive medicine. PATIENT(S): One hundred sixty-eight patients undergoing IVF or intracytoplasmic sperm injection treatment. INTERVENTION(S): Cryopreserved seminal plasma from the patient's partner or sodium chloride (placebo) was injected into the cervix and the posterior fornix of the vagina just after follicle aspiration. MAIN OUTCOME MEASURE(S): Clinical-pregnancy rate. RESULT(S): One hundred sixty-eight patients agreed to participate in the study. Participation was limited to one treatment cycle. Thirty-one patients (18%) were excluded from the study, mainly as a result of canceled embryo transfers. Seventy patients received placebo, and 67 received seminal plasma. The clinical-pregnancy rate was 25.7% (18/70) in the placebo group. The clinical-pregnancy rate in the seminal plasma group reached 37.3% (25/67), corresponding to a relative increase of 45%. CONCLUSION(S): Even though significance was not reached in this pilot study, the data suggest that seminal plasma has the potential to improve pregnancy rate. It is estimated that around 450 patients need to be recruited to reach significance in a multicenter study.

Mice generated by in vitro fertilization exhibit vascular dysfunction and shortened life span

Children conceived by assisted reproductive technologies (ART) display a level of vascular dysfunction similar to that seen in children of mothers with preeclamspia. The long-term consequences of ART-associated vascular disorders are unknown and difficult to investigate in healthy children. Here, we found that vasculature from mice generated by ART display endothelial dysfunction and increased stiffness, which translated into arterial hypertension in vivo. Progeny of male ART mice also exhibited vascular dysfunction, suggesting underlying epigenetic modifications. ART mice had altered methylation at the promoter of the gene encoding eNOS in the aorta, which correlated with decreased vascular eNOS expression and NO synthesis. Administration of a deacetylase inhibitor to ART mice normalized vascular gene methylation and function and resulted in progeny without vascular dysfunction. The induction of ART-associated vascular and epigenetic alterations appeared to be related to the embryo environment; these alterations were possibly facilitated by the hormonally stimulated ovulation accompanying ART. Finally, ART mice challenged with a high-fat diet had roughly a 25% shorter life span compared with control animals. This study highlights the potential of ART to induce vascular dysfunction and shorten life span and suggests that epigenetic alterations contribute to these problems.

Gonadotrophin stimulation for in vitro fertilization significantly alters the hormone milieu in follicular fluid: a comparative study between natural cycle IVF and conventional IVF.

STUDY QUESTION Is the steroid hormone profile in follicular fluid (FF) at the time of oocyte retrieval different in naturally matured follicles, as in natural cycle IVF (NC-IVF), compared with follicles stimulated with conventional gonadotrophin stimulated IVF (cIVF)? SUMMARY ANSWER Anti-Mullerian hormone (AMH), testosterone (T) and estradiol (E2) concentrations are ∼3-fold higher, androstenedione (A2) is ∼1.5-fold higher and luteinizing hormone (LH) is ∼14-fold higher in NC-IVF than in cIVF follicles, suggesting an alteration of the follicular metabolism in conventional gonadotrophin stimulated IVF. WHAT IS KNOWN ALREADY In conventional IVF, the implantation rate of unselected embryos appears to be lower than in NC-IVF, which is possibly due to negative effects of the stimulation regimen on follicular metabolism. In NC-IVF, the intrafollicular concentration of AMH has been shown to be positively correlated with the oocyte fertilization and implantation rates. Furthermore, androgen treatment seems to improve the ovarian response in low responders. STUDY DESIGN, SIZE, DURATION This cross-sectional study involving 36 NC-IVF and 40 cIVF cycles was performed from 2011 to 2013. Within this population, 13 women each underwent 1 NC-IVF and 1 cIVF cycle. cIVF was performed by controlled ovarian stimulation with HMG and GnRH antagonists. PARTICIPANTS/MATERIALS, SETTING, METHODS Follicular fluid was collected from the leading follicles. AMH, T, A2, dehydroepiandrosterone (DHEA), E2, FSH, LH and progesterone (P) were determined by immunoassays in 76 women. Aromatase activity in follicular fluid cells was analysed by a tritiated water release assay in 33 different women. For statistical analysis, the non-parametric Mann-Whitney U or Wilcoxon tests were used. MAIN RESULTS AND ROLE OF CHANCE In follicular fluid from NC-IVF and from cIVF, median levels were 32.8 and 10.7 pmol/l for AMH (P < 0.0001), 47.2 and 18.8 µmol/l for T (P < 0.0001), 290 and 206 nmol/l for A2 (P = 0.0035), 6.7 and 5.6 pg/ml for DHEA (n.s.), 3292 and 1225 nmol/l for E2 (P < 0.0001), 4.9 and 7.2 mU/ml for FSH (P < 0.05), 14.4 and 0.9 mU/ml for LH (P < 0.0001) and 62 940 and 54 710 nmol/l for P (n.s.), respectively. Significant differences in follicular fluid concentrations for AMH, E2 and LH were also found in the 13 patients who underwent both NC-IVF and cIVF when they were analysed separately in pairs. Hormone analysis in serum excluded any relevant impact of AMH, T, A2, and E2 serum concentration on the follicular fluid hormone concentrations. Median serum concentrations were 29.4 and 0.9 mU/ml for LH (P < 0.0001) and 2.7 and 23.5 nmol/l for P (P < 0.0001) after NC-IVF and c-IVF, respectively. Positive correlations were seen for FF-AMH with FF-T (r = 0.35, P = 0.0002), FF-T with FF-LH (r = 0.48, P < 0.0001) and FF-E2 with FF-T (r = 0.75, P < 0.0001). The analysis of aromatase activity was not different in NC-IVF and cIVF follicular cells. LIMITATION, REASONS FOR CAUTION Any association between the hormone concentrations and the implantation potential of the oocytes could not be investigated as the oocytes in cIVF were not treated individually in the IVF laboratory. Since both c-IVF and NC-IVF follicles were stimulated by hCG before retrieval, the endocrine milieu in the natural cycle does not represent the pure physiological situation. WIDER IMPLICATIONS OF THE FINDINGS The endocrine follicular milieu and the concentration of putative markers of oocyte quality, such as AMH, are significantly different in gonadotrophin-stimulated conventional IVF compared with natural cycle IVF. This could be a cause for the suggested lower oocyte quality in cIVF compared with naturally matured oocytes. The reasons for the reduced AMH concentration might be low serum and follicular fluid LH concentrations due to LH suppression, leading initially to low follicular androgen concentrations and then to low follicular AMH production. STUDY FUNDING/COMPETING INTERESTS Funding for this study was obtained from public universities (for salaries) and private industry (for consumables). Additionally, the study was supported by an unrestricted grant from MSD Merck Sharp & Dohme GmbH and IBSA Institut Biochimique SA. The authors are clinically involved in low-dose monofollicular stimulation and IVF therapies, using gonadotrophins from all gonadotrophin distributors on the Swiss market, including Institut Biochimique SA and MSD Merck Sharp & Dohme GmbH. Otherwise, the authors have no competing interests. TRIAL REGISTRATION NUMBER Not applicable.

Modified natural cycle in vitro fertilization an alternative in vitro fertilization treatment with lower costs per achieved pregnancy but longer treatment time

OBJECTIVE To analyze the cost and time requirement per achieved pregnancy in optimized modified natural cycle in vitro fertilization (mNC-IVF) based on a treatment protocol with very few consultations and to compare those with conventional gonadotropin-stimulated aVF (clVF) cycles. STUDY DESIGN Mono centric prospective trial. Eighty infertile patients each received 1 modified mNC-IVF cycle using low doses of the clomiphene citrate. Based on the number of consultations and the clinical pregnancy rate per cycle, the total costs and required time to achieve a pregnancy were analyzed and compared with cIVF. Calculations for cIVF were based on standard therapy protocols and outcomes of European registries. RESULTS Patients (21-42 years old, 35.4 +/- 4.7 years) undergoing mNC-IVF required on average 1.2 consultations before follicle aspiration. Pregnancy rate per transfer and per initiated cycle were 25% and 13.6%, respectively. Multiple pregnancies did not occur. According to the calculations, total costs per pregnancy rate were around 15% lower with mNC-IVF as compared to cIVF. In contrast, time to achieve an equal pregnancy rate was calculated to take around 30% longer with mNC-IVF as compared to cIVF. CONCLUSION mNC-IVF using very low dosages of clomiphene citrate avoids multiple pregnancies and is less expensive but more time consuming per achieved pregnancy when compared to clVF.

The effect of elevated CO2 and increased temperature on in vitro fertilization success and initial embryonic development of single male:female crosses of broad-cast spawning corals at mid- and high-latitude locations

The impact of global climate change on coral reefs is expected to be most profound at the sea surface, where fertilization and embryonic development of broadcast-spawning corals takes place. We examined the effect of increased temperature and elevated CO2 levels on the in vitro fertilization success and initial embryonic development of broadcast-spawning corals using a single male:female cross of three different species from mid- and high-latitude locations: Lyudao, Taiwan (22° N) and Kochi, Japan (32° N). Eggs were fertilized under ambient conditions (27 °C and 500 µatm CO2) and under conditions predicted for 2100 (IPCC worst case scenario, 31 °C and 1000 µatm CO2). Fertilization success, abnormal development and early developmental success were determined for each sample. Increased temperature had a more profound influence than elevated CO2. In most cases, near-future warming caused a significant drop in early developmental success as a result of decreased fertilization success and/or increased abnormal development. The embryonic development of the male:female cross of A. hyacinthus from the high-latitude location was more sensitive to the increased temperature (+4 °C) than the male:female cross of A. hyacinthus from the mid-latitude location. The response to the elevated CO2 level was small and highly variable, ranging from positive to negative responses. These results suggest that global warming is a more significant and universal stressor than ocean acidification on the early embryonic development of corals from mid- and high-latitude locations.

A calcium influx is triggered and propagates in the zygote as a wavefront during in vitro fertilization of flowering plants

In this paper, we report direct measurement of an influx of extracellular Ca2+ induced by gamete fusion in flowering plants. This result was obtained during maize in vitro fertilization with the use of an extracellular Ca2+-selective vibrating probe. Ca2+ influx recorded at the surface of isolated egg cells, with or without adhesion of a male sperm cell, was close to zero and stable over time. Gamete fusion, however, triggered a Ca2+ influx in the vicinity of the sperm entry site with a delay of 1.8 ± 0.6 sec. The Ca2+ influx spread subsequently through the whole egg cell plasma membrane as a wavefront, progressing at an estimated rate of 1.13 μm⋅sec−1. Once established, Ca2+ influx intensities were sustained, monotonic and homogeneous over the whole egg cell, with an average peak influx of 14.92 pmol⋅cm−2⋅sec−1 and an average duration of 24.4 min. The wavefront spread of channel activation correlates well with the cytological modifications induced by fertilization, such as egg cell contraction, and with the cytosolic Ca2+ (c[Ca2+]) elevation previously reported. Calcium influx was inhibited effectively by gadolinium, possibly implicating mechanosensitive channels. Furthermore, artificial influxes created by incubation with Ca2+ ionophores mimicked some aspects of egg activation. Taken together, these results suggest that, during fertilization in higher plants, gamete membrane fusion starts the first embryonic events by channel opening and Ca2+ influx. In turn, c[Ca2+] may work as a trigger and possibly a space and time coordinator of many aspects of egg activation.

Conjoined twins in a monochorionic triplet pregnancy after in vitro fertilization: a case report

Background: Monozygotic monochorionic triplet pregnancy with conjoined twins is a very rare condition and is associated with many complications. Case: In this study, we describe a monochorionic–diamniotic triplet pregnancy after in vitro fertilization with an intracytoplasmic sperm injection. At a gestational age of 6 weeks and 4 days of pregnancy one gestational sac was observed, and at a gestational age of 12 weeks and 2 days, triplets with conjoined twins were diagnosed. After consulting with the parents, they chose fetal reduction of the conjoined twins. Selective feticide was successfully performed by radiofrequency ablation at 16 weeks of pregnancy. Unfortunately, the day after the procedure, the membrane ruptured, and 1 week later, all fetuses and placenta were spontaneously aborted. Conclusion: Monochorionic triplet pregnancy with conjoined twins is very rare. These pregnancies are associated with very serious complications. Intra cytoplasmic sperm injection increases the rate of monozygotic twinning and conjoined twins. Counseling with parents before IVF is very important.

Produção in vitro de embriões de ovinos: uma visão crítica do método e de seu resultado a campo

A metodologia de produção in vitro de embriões de ovinos implica no desenvolvimento de meios de maturação, fertilização e cultivo que permitam aumentar a taxa de clivagem e desenvolvimento, tanto para o investimento biotecnológico em programas comerciais, quanto para sua utilização em clonagem e transgenia dessa espécie animal. Do ponto de vista da pesquisa, os ovócitos podem ser obtidos pelas técnicas de punção e slicing a partir de ovários oriundos de matadouros, ou através de aspiração folicular por laparoscopia. Como vantagem, este método permite o uso de uma mesma doadora estimulada hormonialmente em intervalos periódicos, mantida sob rigoroso controle sanitário, o que é de vital importância para a produção de biofármacos em programas que utilisem os ovinos como modelo biológico. Por outro lado, em nosso país a demanda pela multiplicação de animais de alto valor genético, seja pela produtividade ou pelo elevado valor comercial dos mesmos, impõe o desenvolvimento, adaptação e otimização das diferentes metodologias desenvolvidas ao longo dos ultimos anos em laboratórios de referência mundiais. Nesse contexto, cresce de importância o perfeito conhecimento da fisiologia dessa espécie e das raças criadas em nosso país, e da problemática da produção in vitro de seus embriões. Respeitando essas premissas, gerar o desenvolvimento de protocolos que permitam não apenas aumentar a população de ovócitos passíveis de maturação in vitro, mas de sua competência ao desenvolvimento ao estágio de blastocisto, ou, alternativamente, sua transferência a receptoras em estágios precoces do desenvolvimento, evitando assim as conhecidas perdas durante o desenvolvimento in vitro, e o baixo percentual de gestações que chegam a termo, com cordeiro saudáveis. Trata-se de um desafio, que já apresenta os primeiros resultados em nosso país, tanto na produção comercial de embriões produzidos in vitro, quanto em programas de clonagem e transgenia.

Influence of Caffeine and Chondroitin Sulfate on Swine Sperm Capacitation and In Vitro Embryo Production

Background: The establishment of an in vitro production (IVP) of embryo in swine allows the generation of embryos with the same quality as in vivo produced embryos with less costs and time. In order to achieve successful fertilization under normal circumstances in vivo, mammalian spermatozoa must first undergo capacitation and then acrosome reaction. The purpose of this study was compared the efficacious of IP/CFDA fluorescence and Coomassie Blue G (CB) staining to detect capacitated sperm cells in refrigerated and fresh semen. Morever, it was investigated the efficacious of caffeine and chondroitin sulphate to promote in vitro sperm capacitation and in vitro embryo produced (IVP) of swine embryos. Materials, Methods & Results: A sperm-rich fraction from ejaculate was obtained using the gloved-hand method and the gel-free fraction was separated using sterile gauze. The semen was diluted in BTS at a final concentration of 1.5 x 10(8) cells/mL. The sperm suspension was incubated for 2 h at 25 degrees C, refrigerated and maintained for 1 h at 15-18 degrees C (refrigerated group) or used immediately (fresh group). Sperm capacitation was assessed by IP/CFDA fluorescence and CB staining for both fresh and refrigerated semen. For PI/CFDA evaluation, a final solution containing 1.7 mM formaldehyde, 7.3 mM PI and 20 mM CFDA in 950 mu L saline was prepared. In the dark, 40 mu L PI/CFDA final solution was added to 10 mu L semen and after 8 min, slides were analyzed on epifluorescence microscopy. For CB evaluation, sperm cells were fixed in 4% paraformaldehyde for 10 min and centrifuged twice at 320 x g in ammonium acetate pH 9 for 8 min. A smear was made and stained with 2.75 mg/mL CB in solution containing 12.5% methanol, 25% glacial acetic acid and 62.5% water, for 2 min. The smear was washed in running water, air dried and sealed with Permount (R), diluted 2:1 in xilol to avoid staining oxidation. Our results showed that refrigeration did not affect sperm capacitation and comparing staining methods, the PI/CFDA combination was more efficient to detect capacitated sperm, when compared to CB staining. In experiment 2, we evaluated the effect of different incubation time (1 - 5 h) with chondroitin sulfate and caffeine on sperm capacitation. For in vitro fertilization, oocytes were obtained from slaughterhouse ovaries. Oocytes with a thick and intact cumulus oophurus layer and cytoplasm with homogenous granules were selected for in vitro maturation for 44 h. According to the results of experiment 2, it was used for in vitro fertilization refrigerated semen was capacitated with 50 mu g/mL chondroitin sulfate for 2 h or capacitated with 5 mu g/mL caffeine for 3 h. Six hours after insemination, cumulus oophorus cells were mechanically removed and oocytes were washed and incubated in microdrops of culture medium. Embryo development after fertilization with sperm capacitated with caffeine or chondroitin sulfate was evaluated on days 3, 5 and 7 of culture. No differences were observed in days 3 or 5 of in vitro culture. However, it was observed an increase on blastocyst rate on Day 7 of culture when caffeine was used as the capacitor agent. Discussion: Molecular basis of sperm capacitation is still poor understood. Sperm capacitation can occur in vitro spontaneously in defined media without addition of biological fluids. We observed that sperm capacitation increased as incubation period enlarged and it was observed using Coomassie blue G and PI/CFDA for fresh semen and for refrigerated semen. It can be concluded that the cooling of semen did not change their pattern of sperm capacitation and this is best assessed by IP/CFDA than by CB. In addition to the use of caffeine in sperm capacitation produces more blastocysts than the chondroitin sulfate after in vitro fertilization.

Gene silencing during development of in vitro-produced female bovine embryos

In early development, female embryos (XX) produce twice the transcripts of X-linked genes compared with male embryos (XY). During the course of development, inactivation of the X chromosome equilibrates gene dosage, making the development of female embryos viable. Moreover, the biotechnologies used for producing embryos in vitro seem to work better with male embryos, making it easier for them to reach the blastocyst stage and allow for complete gestation. We investigated the expression of three X-linked genes that are involved in development, XIST, G6PD, and HPRT, and of the transcript interferon-tau, in male and female bovine blastocysts produced by nuclear transfer (NT) and by in vitro fertilization (IVF). Oocytes that had been matured in vitro were enucleated and reconstructed with somatic cells from adult animals at 18 h post-maturation. After fusion (two pulses of 2.25 kv/cm) and chemical activation (5.0 mu M ionomycin for 5 min and 2.0 mM 6-DMAP for 3 h), the oocytesomatic cell units were cultivated in CR2 with a monolayer of granulosa cells at 38.8 degrees C, in a humidified 5% CO(2) atmosphere. IVF embryos were inseminated, after centrifugation in a Percoll gradient, with 2 x 10(6) sperm/mL TALP medium supplemented with BSA and PHE and cultivated under the same conditions as the cloned embryos. We used real-time PCR to analyze the gene expression of individual blastocysts compared to expression of the housekeeping gene, GAPDH. The gene XIST was expressed in female embryos and not in male embryos produced by IVF, though it was expressed at low levels in male embryos produced by NT. Unlike previous reports, we found lower levels of the transcript of G6PD in females than in males, suggesting double silencing or other mechanisms of control of this gene. Female embryos produced by IVF expressed the HPRT gene at a higher level than female embryos produced by NT, suggesting that gene silencing proceeds faster in NT-produced female embryos due to ""inactivation memory"" from the nucleus donor. In conclusion, male and female embryos express different levels of X-chromosome genes and failures of these genes that are essential for development could reduce the viability of females. Nuclear transfer can modify this relation, possibly due to epigenetic memory, leading to frequent failures in nuclear reprogramming.

Imprinted gene expression in in vivo- and in vitro-produced bovine embryos and chorio-allantoic membranes

Cloning by nuclear transfer is often associated with poor results due to abnormal nuclear reprogramming of somatic donor cells and altered gene expression patterns. We investigated the expression patterns of imprinted genes IGF2 and IGF2R in 33- to 36-day bovine embryos and chorio-allantoic membranes derived from in vivo- and in vitro-produced embryos by somatic cell nuclear transfer (SCNT), parthenogenetic activation, and in vitro fertilization (IVF). There was a lower IGF2 expression rate in the SCNT (0.19) and parthenogenetic (0.02) groups when compared to in vivo and IVF embryos (2.01; P < 0.05). In the chorio-allantoic membranes, IGF2 showed a baseline expression pattern (P < 0.05) in parthenotes (0.001) when compared to in vivo, IVF (3.13), and SCNT (0.98) groups. IGF2R was less expressed (P < 0.05) in SCNT chorio-allantoic membranes (0.25) when compared to the in vivo group. The low expression of IGF2 in parthenogenetic embryos and chorio-allantoic membranes confirms its imprinted status in cattle. Alterations in the relative frequency of IGF2 and IGF2R transcripts were observed in SCNT-derived bovine embryos and chorioallantoic membranes, respectively, supporting the hypothesis that abnormalities in the expression of imprinted genes are causes of the low efficiency of SCNT procedures in this species.

Biological Effects and Dose-Response Assessment of Diesel Exhaust Particles on In Vitro Early Embryo Development in Mice

An increased risk of early pregnancy loss in women briefly exposed to high levels of ambient particulate matter during the preconceptional period was recently observed. The effects of this exposure on early embryo development are unknown. This study was designed to assess the dose-response and biological effects of diesel exhaust particles (DEP) on in vitro embryo development using the in vitro fertilization (IVF) mouse model. Zygotes obtained from superovulated mice after IVF were randomly cultured in different DEP concentrations (0, 0.2, 2, and 20 mu g/cm(2)) for 5 days and observed for their capacity to attach and develop on a fibronectin matrix until day 8. Main outcome measures included blastocyst rates 96 and 120 h after insemination, hatching discriminatory score, total cell count, proportion of cell allocation to inner cell mass (ICM) and trophectoderm (TE), ICM morphology, attachment rate and outgrowth area, apoptosis and necrosis rates, and Oct-4 and Cdx-2 expression. Multivariate analysis showed a negative dose-dependent effect on early embryo development and hatching process, blastocyst cell allocation, and ICM morphology. Although blastocyst attachment and outgrowth were not affected by DEP, a significant impairment of ICM integrity was observed in day 8 blastocysts. Cell death through apoptosis was significantly higher after DEP exposure. Oct-4 expression and the Oct-4/Cdx-2 ratio were significantly decreased in day 5 blastocysts irrespective of DEP concentration. Results suggest that DEP appear to play an important role in disrupting cell lineage segregation and ICM morphological integrity even at lower concentrations, compromising future growth and viability of the blastocyst.

Leuprolide acetate reduces both in vivo and in vitro ovarian steroidogenesis in infertile women undergoing assisted reproduction

Despite the probable inhibitory effects of GnRH analogues on ovarian steroidogenesis in vitro, their association with assisted reproduction protocols shows favorable results. This suggests that there are important differences in the behaviors of these drugs when administered in vivo versus in vitro. To clarify these differences, this study was designed to analyze the effect of leuprolide acetate (LA) on ovarian steroidogenesis in women undergoing In Vitro Fertilization (IVF). A prospective, randomized open label study was conducted on 14 women (26-35 years): seven receiving only gonadotrophins (Group 1) and seven receiving gonadotrophin plus LA at 1mg/day (Group 2). The LA in vivo effect was determined with serum and follicular fluid (FF) samples and via luteinized granulosa cell cultivation (GCC), where cells were obtained during oocyte retrieval after ovarian hyperstimulation. In vitro analysis was performed via addition of LA to GCC only for Group 1 (without LA) at progressively higher concentrations (0, 10(-12), 10(-9) and 10(-6) M). In vivo, the main observation was a reduction in androgen production in Group 2, represented by lower androstenedione production in FF (G1 = 6479 +/- 3458; G2 = 3021 +/- 1119 ng/ml; p = 0.04) and a lower testosterone peak in GC at 96 h (G1 = 0.64 +/- 0.12 ng/ml; G2 = 0.50 +/- 0.19ng/ml; P = 0.02), but a higher fertilization rate (G1 = 67%; G2 = 83%; p = 0.009). in vitro, testosterone, estradiol and progesterone were also reduced by LA, even though this reduction occurred for progesterone only at the highest LA dosage (10(-6) M; 606.0 +/- 114.3 ng/ml versus 1524.0 +/- 246.5 ng/ml; p=0.02). Results show that LA reduces ovarian steroidogenesis in vivo by essentially inhibiting androgen synthesis; whereas, in vitro, ovarian steroidogenesis is reduced overall. (C) 2008 Elsevier Inc. All rights reserved.

Apoptotic and developmental effects of bovine Herpesvirus type-5 infection on in vitro-produced bovine embryos

Bovine Herpesvirus type-5 (BoHV-5), which is potentially neuropathogenic, was recently described to be related with reproductive disorders in cows. The objective was to elucidate mechanisms involved in propagation of BoHV-5 in embryonic cells. For this purpose, bovine embryos produced in vitro were assayed for apoptotic markers after experimental infection of oocytes, in vitro fertilization, and development. Host DNA fragmentation was detected with a TUNEL assay, expression of annexin-V was measured with indirect immunofluorescence, and viral DNA was detected with in situ hybridization. Infective BoHV-5 virus was recovered from embryos derived from exposed oocytes after two consecutive passages on Madin-Darby bovine kidney (MDBK) cells. The viral DNA corresponding to US9 gene, localized between nucleotides 126243 to 126493, was detected in situ and amplified. There was no significant difference between the ratio of TUNEL stained nuclei and total cells in good quality blastocysts (0.87 +/- 0.05, mean SD), but there were differences (P < 0.05) between infected (0.18 +/- 0.05) and uninfected blastocysts (0.73 +/- 0.07). The Annexin-V label was more intense in uninfected embryos (0.79 +/- 0.04; P < 0.05). The quality of infected and uninfected embryos was considered equal, with no significant effect on embryonic development. In conclusion, we inferred that BoHV-5 infected bovine oocytes, replicated, and suppressed some apoptotic pathways, without significantly affecting embryonic development. (C) 2010 Elsevier Inc. All rights reserved.