Effects of sera, hormones and granulosa cells added to culture medium for in-vitro maturation, fertilization, cleavage and development of bovine oocytes

Sera (fetal calf serum: FCS; and oestrous cow serum: ECS), hormones (2.5 FSH micrograms/ml + 5 micrograms LH/ml + 1 microgram oestradiol/ml) and granulosa cells (5 x 10(6)/ml) were added to culture medium to determine the frequencies of in-vitro maturation, fertilization, cleavage (2- to 8-cell) and development into blastocysts of bovine follicular oocytes. The maturation rates after 24 h in culture were not significantly different among the three factors tested (56-72%). The fertilization rates were significantly affected by serum type and the addition of granulosa cells. FCS gave significantly higher rates of fertilization (57-71%) than did ECS (34-52%), but the proportions of polyspermic fertilization were significantly higher in the former (8-19%) than in the latter (2-3%). The addition of hormones did not affect fertilization, cleavage and development. Neither type of serum affected cleavage and development. The highest rates of blastocyst formation were obtained when granulosa cells alone were added (FCS, 17%; ECS, 16%). The cell numbers of the blastocysts obtained were 100-150, similar to those of blastocysts developed in vivo. Transfer of 6 blastocysts to 3 cows resulted in 1 pregnancy. The present results indicate that the co-culture with granulosa cells is the most important factor for in-vitro fertilization to development into blastocysts of bovine oocytes matured in vitro.     

Follicular fluid adrenomedullin concentrations in spontaneous and stimulated cycles: relationship to ovarian function and endothelin-1 and nitric oxide

The objective of this study was to determine concentration of adrenomedullin (AM) in follicular fluid and whether a correlation exists between AM and nitric oxide (NO) or endothelin-1 (ET-1) levels in follicular fluid, serum 17beta-estradiol or other parameters of ovarian function in spontaneous and gonadotrophin stimulated ovarian cycles. Follicular fluid samples were obtained at oocyte retrieval from 50 women who underwent an in vitro fertilization (IVF) program: 40 undergoing ovarian hyperstimulation with recombinant FSH and 10 had spontaneous ovarian cycles. AM, ET-1, and NO were detected in all of the follicular fluid samples and their concentrations were similar in spontaneous and stimulated cycles. In patients undergoing ovarian stimulation, follicular fluid AM levels correlated with serum 17beta-estradiol concentration. No correlation was found between follicular AM concentration and parameters of ovarian function. Similarly, no relationship was observed between ET-1, NO, and AM follicular fluid concentrations in either spontaneous or stimulated cycles.This study suggests a possible regulatory effect of the sexual hormones on AM production by the ovary during the ovulatory process. The site of AM secretion and its function (if any), however, remain to be established.     

Ovarian Filariasis: Diagnosis by detection of microfilariae in follicular fluid,a case report

Filariasis is a common parasitic infection in India. It is rare to find neglected cases of Filariasis nowadays. We reported the presence of microfilaria species in the follicular fluid of an egg donor undergoing an ovum pick up procedure. She was a 23-year-old egg donor who underwent stimulation using the GnRH antagonist protocol. Antagonist protocol is one of the standard protocols used for controlled ovarian hyperstimulation as a part of the IVF/ICSI(in-vitro fertilization / intracytoplasmic sperm injection) procedure where GnRH antagonist (cetrorelix) is used to suppress the endogenous LH surge. Her baseline investigations were normal, with no significant history suggestive of any worm infestations. During the ovum pickup procedure, follicular fluid revealed the presence of worm-like structures suggestive of larvae of some parasites. The follicular fluid was sent to the microbiology department along with the blood sample to confirm the parasite species. The parasite was found to be the larvae of W. Bancroft. The oocytes were of poor quality and were discarded. The patient was treated with Diethylcarbamazine citrate. There are so many reports about scrotal Filariasis, but rare literature quotes ovarian Filariasis.     

Insulin and insulin-like growth factor I in follicular fluid after induction of ovulation in women undergoing in vitro fertilization.

This study was undertaken to evaluate the relationship between concentrations of insulin and insulin-like growth factor I (IGF-I) in follicular fluid and fertilization and cleavage of human oocytes fertilized in vitro. The concentration of oestradiol, progesterone, luteinizing hormone, follicle-stimulating hormone, testosterone, insulin and IGF-I was determined in 36 follicular fluids, free of visible blood contamination and containing mature oocyte-corona-cumulus complexes, obtained from 12 women undergoing in vitro fertilization. Follicular development was induced by clomiphene citrate and human menopausal gonadotrophin, and follicular aspiration was performed 35 h after an ovulatory dose of human chorionic gonadotrophin. Concentrations of IGF-I were significantly higher in follicular fluids associated with mature oocytes that fertilized and cleaved, than in follicular fluid associated with mature oocytes that did not fertilize (P < 0.001). There was no difference in the concentration of insulin between follicular fluids from which fertilized oocytes were obtained and those with oocytes that remained unfertilized. No significant correlations were found between rates of embryo cleavage, concentrations of insulin and IGF-I. Multiple linear regression analysis demonstrated that the concentrations of IGF-I in follicular fluid were predicted statistically by a negative regression coefficient for the concentration of testosterone, and by a positive regression coefficient for the concentration of progesterone in follicular fluid. No candidate variable was included in the model to predict concentrations of insulin. These data suggest an important role for IGF-I in the mature follicle.     

Formation of lipoidal steroids in follicular fluid

The presence of high levels of lipoidal pregnenolone in follicular fluid has recently been established although no evidence has been presented concerning its possible origin. The following investigation focuses on the enzymatic conversion of non-conjugated steroids into their lipoidal derivatives in preovulatory follicular fluid obtained from women undergoing in vitro fertilization. Our observations indicated that pregnenolone, an important precursor steroid, was acylated at a similar rate as cholesterol in follicular fluid. Similar studies were subsequently conducted with serum obtained from a pool of normal women and women undergoing follicular stimulation which showed little difference to the results obtained in follicular fluid. Further studies using dehydroepiandrosterone, androst-5-ene-3 beta,17 beta-diol, estradiol and dihydrotestosterone were were also performed to monitor their respective lipoidal conversion percentages in follicular fluid which revealed a marked difference of conversion rates between steroids. The indirect identification of the lipoidal pregnenolone derivatives formed in follicular fluid was also conducted by incubating radiolabelled pregnenolone in follicular fluid. The fatty acid components of the resulting lipoidal pregnenolone derivatives showed a marked resemblance to those of cholesteryl esters formed in plasma by the enzymatic activity of lecithin:cholesterol acyltransferase. The pregnenolone derivatives were comprised predominantly of unsaturated fatty acids such as linoleate, palmitoleate, oleate, linolenate and arachidonate while saturated fatty acids, namely palmitate, constituted 20% of the total lipoidal pregnenolone.     

Effects of Multivitamin/Mineral Supplementation on Trace Element Levels in Serum and Follicular Fluid of Women Undergoing in Vitro Fertilization (IVF)

We investigated effects of multivitamin/mineral supplementation on element levels in serum and follicular fluid of women undergoing IVF. We used three groups in this study. The first group was used as an age-matched and nonpregnant control (n = 13). Group 2 (n = 30) constituted the IVF group and women in the third group who were undergoing IVF also received a multivitamin/mineral tablet daily for 45 days. Follicular fluid and serum selenium and zinc levels and follicular fluid copper levels were lower in IVF patients than in controls although follicular fluid aluminum and iron levels were higher in IVF patients than in controls. However, follicular fluid and serum aluminum, copper, zinc and selenium levels, and serum magnesium levels were higher in the multivitamin/mineral group than in the IVF group although follicular fluid iron levels were lower in the multivitamin/mineral group than in the IVF group. In conclusion, we observed that copper, zinc, and selenium in serum and follicular fluid decreased in women undergoing IVF. Multivitamin/mineral supplementation in serum and follicular fluid of women undergoing IVF normalized the trace element levels.     

Microorganisms within Human Follicular Fluid: Effects on IVF

Our previous study reported microorganisms in human follicular fluid. The objective of this study was to test human follicular fluid for the presence of microorganisms and to correlate these findings with the in vitro fertilization (IVF) outcomes. In this study, 263 paired follicular fluids and vaginal swabs were collected from women undergoing IVF cycles, with various causes for infertility, and were cultured to detect microorganisms. The cause of infertility and the IVF outcomes for each woman were correlated with the microorganisms detected within follicular fluid collected at the time of trans-vaginal oocyte retrieval. Microorganisms isolated from follicular fluids were classified as: (1) ‘colonizers’ if microorganisms were detected within the follicular fluid, but not within the vaginal swab (at the time of oocyte retrieval); or (2) ‘contaminants’ if microorganisms detected in the vagina at the time of oocyte retrieval were also detected within the follicular fluid. The presence of Lactobacillus spp. in ovarian follicular fluids was associated with embryo maturation and transfer. This study revealed microorganisms in follicular fluid itself and that the presence of particular microorganisms has an adverse affect on IVF outcomes as seen by an overall decrease in embryo transfer rates and pregnancy rates in both fertile and infertile women, and live birth rates in women with idiopathic infertility. Follicular fluid microorganisms are a potential cause of adverse pregnancy outcomes in IVF in both infertile women and in fertile women with infertile male partners.     

The interaction between follicular fluid total antioxidant capacity,infertility and early reproductive outcomes during in vitro fertilization

Abstract

Introduction: The objective of this study was to determine whether the relationship between antioxidant capacity of follicular fluid and early reproductive outcomes is influenced by the cause of infertility, polycystic ovarian morphology, age and smoking.

Patients and methods: This was a prospective, cross-sectional study performed in an assisted conception unit and a teaching hospital. The study cohort was 34 women undergoing IVF treatment. Interventions included total antioxidant capacity (TAC) measured using ferric reducing/antioxidant power assay in 303 follicular fluid samples. The main outcome measures were follicular fluid TAC, percentage TAC loss after 72 h and early reproductive outcomes.

Results: Follicular TAC was elevated in women with infertility of 'unexplained' (UE) or tubal factor (TF) aetiology, relative to those with male factor (MF) infertility, when reproductive outcomes were positive but not when they were negative. In the TF and UE groups, low TAC was associated with ovum fertilization incompetence, whereas TAC was comparable irrespective of embryo viability. Unexplained infertility was associated with significantly elevated follicular TAC. Among women with polycystic ovaries, fertilization incompetence was associated with elevated TAC; the opposite was true in women with normal ovaries. Follicular fluid 72-h TAC consumption > 20% was associated with poorer reproductive performance.

Conclusions: The follicular fluid pro-oxidant-antioxidant balance required for conception in women undergoing IVF is related to the aetiology of infertility, age, the presence of polycystic ovary morphology and smoking.     

Characterization of Chemical Contamination in Shallow-Water Estuarine Habitats of an Industrialized River. Part 1: Organic Compounds

The lower six miles of the tidal portion of the Passaic River (Study Area) has been heavily industrialized since the mid-1800s. The objectives of this study were to: quantify the present extent and magnitude of chemical contamination in surface sediments from the Study Area; evaluate the contamination in the Study Area relative to a reference area and surrounding regional waterways; assess the potential for adverse effects to aquatic organisms; and identify spatial gradients in concentrations that may indicate potential point-sources of chemicals. Because existing ecological communities center primarily on intertidal mudflats in the Study Area, composite surface sediment was collected from 15 mudflats to generate a realistic assessment of fish and wildlife exposure to sediment contaminants. This study showed that present concentrations of organic contaminants are elevated throughout the Study Area and are generally higher than concentrations in the reference area and regional waterways. A screening-level analysis showed that there is potential for adverse effects to sediment-associated organisms from a number of chemicals. No spatial trends were apparent for most compounds evaluated, consistent with the presence of multiple sources of chemicals in the Study Area. The exception is semivolatile compounds, which appear to be localized to specific mudflats, indicating potential point-sources of contamination for these chemicals.     

Assessment of human contamination of estrogenic endocrine-disrupting chemicals and their risk for human reproduction

There is broad human exposure to estrogenic endocrine-disrupting chemicals (EDCs), but the data sets that exist are primarily for various environmental media such as food and water rather than the most relevant internal exposure. We have detected various kind of EDC contamination in humans including dioxin and bisphenol A (BPA) widely used for the production of plastic products. BPA was present in serum and follicular fluid at approximately 1–2 ng/ml, as well as in fetal serum and full-term amniotic fluid, confirming passage through the placenta. An approximately five-fold higher concentration, 8.3 ± 8.7 ng/ml, was revealed in amniotic fluid at 15–18 weeks of gestation, compared to other fluids showing increased exposure at the critical developmental period in humans. Interestingly, serum BPA concentrations were significantly higher in normal men and in women with polycystic ovary syndrome (PCOS) compared with normal women possibly due to differences in the androgen-related metabolism of BPA. These findings may provide some insight into the metabolism of EDCs in human and the pathophysiology of endocrine disorders such as PCOS. Dioxin contamination in relationship to development of endometriosis is also discussed.     

Production of lysophosphatidic acids by lysophospholipase D in human follicular fluids of In vitro fertilization patients.

Lysophosphatidic acids (LPAs) are known to be normal constituents of mammalian serum, and they mimic some biological effects of the serum. We previously reported that lysophospholipase D (LPLD) was involved in the accumulation of LPAs in incubated rat plasma and serum. In this study we detected, by gas-liquid chromatography, various molecular species of LPA in follicular fluids collected from women programmed for in vitro fertilization. When the follicular fluid was incubated at 37 degrees C for 48 h, persistent increases in the amounts of LPAs were observed concomitant with decreases in the amounts of the corresponding lysophosphatidylcholines (LPCs), although the concentrations of saturated LPCs increased in the first 6 h of incubation. These results suggest that human follicular fluid has LPLD activity, and this was confirmed by experiments with follicular fluids mixed with an exogenous radioactive LPC. The LPLD showed preference for unsaturated over saturated LPCs, similar to plasma LPLD, indicating that it originated from the circulation.     

The determination of 11 beta-hydroxyandrostenedione in human follicular fluid and plasma

The development of a chromatographic/immunoassay method is presented for the measurement of 11 beta-hydroxyandrostenedione (11 beta-OH-A4) in ovarian follicular fluid (FFL) and plasma from women undergoing embryo transfer for in vitro fertilization. This method incorporates high-performance liquid chromatography (HPLC) and permits the simultaneous measurement of other steroids from a single sample in order to assess the intraovarian environment. Authenticity of 11 beta-OH-A4 in follicular fluid was confirmed using selected ion monitoring (SIM) gas chromatography/mass spectrometry (GC/MS). Our results demonstrate a mean concentration of 18.6 nmol/l in follicular fluid compared with 3.2 nmol/l in plasma. The origin of 11 beta-OH-A4 in follicular fluid requires further investigation but these findings supports the hypothesis of ovarian 11 beta-hydroxylase activity on C19 steroids.     

Modulation of spermatozoa and zona pellucida properties by the soluble acrosome reaction-inducing factor of the ovulated egg-cumulus complex

Three sources of hamster periovulatory fluids (± heat inactivation at 56°C), with bovine serum albumin (BSA) as control, were tested for effects on penetration of three classes of eggs by hamster sperm precapacitated in BSA. These fluids were a soluble extract of cumulus oophorus fluid (COF) from the ovulated hamster egg-cumulus complex, serum, and follicular fluid. Egg types were ovulated, salt-stored (ovulated), and follicular. In both COF and serum, there were significant differences among egg types in mean penetration, and significant effects of fluid addition. In contrast, there was no effect of follicular fluid and no differences between follicular and stored eggs. For the follicular eggs (combined data, normalized, ranked), patterns of response to the three factors (± heating) were different: only unheated COF and heated serum increased penetration significantly above BSA control levels (average rank 20.2, 41.4, 38, for BSA, COF (unheated), serum (heated), respectively). This indicated that the active component in COF was heat labile, not present in either serum or follicular fluid, and, therefore, of oviductal origin. Oviduct and/or COF exposure of eggs and sperm was tested for effects as an acrosome reaction inducing factor (ARIF) for acrosome reactions (AR; zonabound and free-swimming sperm) and on sperm:zona binding and penetration. The COF ARIF for free-swimming sperm AR was heat stable. Penetration of follicular eggs increased after incubation in COF prior to sperm addition, but a greater response occurred when COF was added to eggs with sperm. In kinetic experiments, 25 min following sperm attachment, follicular eggs had lost 41% of initially bound sperm, vs. 23% for ovulated eggs, and had only 16 AR sperm/egg, vs. 26 for ovulated. Follicular eggs incubated in COF (then washed three times) had the same number of bound AR sperm as ovulated eggs. Acid solubilized zona pellucida (ASZP) from ovulated eggs was more effective as an ARIF per zona than ASZP from follicular eggs. Zonae of follicular eggs, as eivdenced by dissolution times in β-mercaptoethanol (β-MEOH), were not “harder” than those of ovulated eggs. There were differences in lectin binding antigens on zonae of both fresh and stored, follicular and ovulated, eggs. We conclude that multiple biological factors orchestrate sperm:egg interactions in the ampulla. Our data are consistent with the presence of at least three effective components: (1) the oviductal lectin-binding antigen (ZPO or oviductin), (2) an additional heat-labile component, and (3) the heat-stable ARIF for free-swimming sperm. © 1994 Wiley-Liss, Inc.     

Presence of fatty acid esters of pregnenolone in follicular fluid from women undergoing follicle stimulation

In the following investigation the presence of lipoidal pregnenolone derivatives in the preovulatory follicular fluid obtained from women undergoing in vitro fertilization was established. Concentrations of lipoidal pregnenolone proved to be at least twofold greater than those of the unconjugated counterpart. Indirect identification of these lipoidal pregnenolone derivatives was achieved by comparing the C-18 column, thin-layer silica gel (TLC), high performance liquid chromatography (HPLC), and gas chromatography/mass spectrometry (GC/MS) chromatographic properties of the endogenous lipoidal pregnenolone derivatives in follicular fluid with those of synthetic acyl pregnenolone esters. Lipoidal pregnenolone derivatives recovered after HPLC subfractionation were treated with alkali to hydrolyze the acyl group thus liberating nonconjugated pregnenolone. Concentrations of this steroid were then measured using radioimmunoassay upon which analysis of HPCL and gas chromatograms permitted the calculation of the individual pregnenolone ester contributions within the samples. Five lipoidal pregnenolone derivatives constituted more than 90% of the total lipoidal pregnenolone concentration observed, these derivatives being: pregnenolone oleate (30.7%), linoleate (20.7%), palmitate (20.1%), linolenate (14.8%), and palmitoleate (7.1%).     

A detailed analysis of early events during in-vitro fertilization of bovine follicular oocytes

Bovine follicular oocytes collected at slaughter were matured and fertilized in vitro with in-vitro capacitated spermatozoa. Analysis of 621 penetrated ova fixed at various times after in-vitro insemination led to definition of 6 stages of early development. A time sequence for sperm penetration, sperm head decondensation, male pronucleus formation, the activation of second meiotic division, female chromosome decondensation and pronucleus development was established. First sperm penetration into the ooplasm was recorded 6 h after insemination; 1-2 h was required for the sperm head to decondense and another 4-6 h to develop into the opposing pronucleus stage. Synkaryosis and first cleavage occurred 28 h after fertilization. Examination of the early stages revealed four types of abnormalities, i.e. polyspermy, polygyny, asynchrony between male and female pronucleus development, and preactivation of cytokinesis.     

Fertilization in vitro of bovine oocytes after various sperm procedures

Fertilization in vitro of bovine follicular oocytes cultured in vitro was attempted after various procedures on frozen-thawed bull spermatozoa. The frozen-thawed semen was diluted at 1 : 15 and treated with one of eight methods as follows: 1) no washing, 2) washing, 3) passing through a glass wool column, 4) washing and bovine follicular fluid (BFF), 5) Ham's F-12 based medium and BFF (1 : 1), 6) BFF only, 7) a high ionic strength (HIS) treatment and the medium, or 8) HIS treatment and BFF. A total of 766 oocytes was examined for the identification of fertilization (the presence of the pronuclei and a sperm midpiece in the oocyte cytoplasm and the second polar body) and cleavage at 24 h after insemination. The sperm procedures by BFF treatment with or without washing showed significantly higher rates of fertilization (P<0.05) than the other methods tested, except after HIS treatment. The highest fertilization rate (46.2%) was obtained by the treatment with BFF only. The results indicate that BFF could favorably affect capacitation of frozen-thawed bull spermatozoa and its subsequent fertilization in vitro.     

In vitro fertilization of in vitro-matured equine oocytes: effect of maturation medium,duration of maturation,and sperm calcium ionophore treatment,and comparison with rates of fertilization in vivo after oviductal transfer

Three experiments were conducted to evaluate the effect of oocyte and sperm treatments on rates of in vitro fertilization (IVF) in the horse and to determine the capacity of in vitro-matured horse oocytes to be fertilized in vivo. There was no effect of duration of oocyte maturation (24 vs. 42 h) or calcium ionophore concentration during sperm capacitation (3 microM vs. 7.14 microM) on in vitro fertilization rates. Oocytes matured in 100% follicular fluid had significantly higher fertilization (13% to 24%) than did oocytes matured in maturation medium or in 20% follicular fluid (0% to 12%; P < 0.05). There was no significant difference in fertilization rate among 3 sperm treatments utilizing 7.14 microM calcium ionophore (12% to 21%). Of in vitro-matured oocytes recovered 40-44 h after transfer to the oviducts of inseminated mares, 77% showed normal fertilization (2 pronuclei to normal cleavage). Cleavage to 2 or more cells was seen in 22% of oocytes matured in follicular fluid and 63% of oocytes matured in maturation medium; this difference was significant (P < 0.05). We conclude that in vitro-matured horse oocytes are capable of being fertilized at high rates in the appropriate environment and that in vitro maturation of oocytes in follicular fluid increases fertilization rate in vitro but reduces embryo development after fertilization in vivo. Further work is needed to determine the optimum environment for sperm capacitation and IVF in the horse.     

Induction of the acrosome reaction in human spermatozoa by a fraction of human follicular fluid

Human ejaculated spermatozoa were washed through a Percoll gradient, preincubated for 10 hr in a defined medium containing serum albumin, and then induced to undergo rapid acrosome reactions by addition of human follicular fluid or a Sephadex G-75 column fraction of the fluid. Induction by follicular fluid did not occur when the spermatozoa were preincubated for only 0 or 5 hr. The reactions were detected by indirect immunofluorescence using a monoclonal antibody directed against the human sperm acrosomal region. The percentage of acrosomal loss counted by transmission electron microscopy agreed with that counted by immunofluorescence. The apparent molecular weight of the Sephadex G-75 fraction containing the peak of acrosome reaction-inducing activity was 45,000 ± 4,200 (SD). The occurrence of physiological acrosome reactions was supported by: assessing motility (no significant loss of motility occurred during the treatment period when sperm were preincubated with bovine serum albumin), transmission electron microscopy (the ultrastructural criteria for the acrosome reaction were met), and zona-free hamster oocyte binding and penetration (spermatozoa pretreated with the active fraction of follicular fluid, then washed and incubated with oocytes, showed significantly greater binding to and penetration of oocytes). The stimulation of the acrosome reaction by follicular fluid is apparently not due to blood serum contamination; treatment of preincubated spermatozoa with sera from the follicular fluid donors had no effect on the spermatozoa. The nature of the active component(s) in that fraction is currently being investigated.     

Influence of temperature and gas atmosphere on in-vitro fertilization and embryo development in domestic cats

The influence of culture temperature and gas atmosphere on in-vitro fertilization and embryo development was examined in the domestic cat. In Exp. 1, eggs were fertilized and cultured in 5% CO2 in air at 37, 38 or 39 degrees C. Experiment 2 evaluated the effects of 5% CO2 in air; 5% CO2, 5% O2 and 90% N2; and 10% CO2 in air. Fertilization (cleavage) and development to the morula/blastocyst stage were not influenced (P greater than 0.05) by variations in temperature and gas composition. Despite changing these culture conditions, egg cleavage averaged approximately 75% and greater than 80% of the 2-cell embryos proceeded to morulae in vitro. However, the partial in-vitro morula-to-blastocyst developmental block normally observed in this species was not removed.     

Lead concentrates in ovarian follicle compromises pregnancy

Following absorption, lead can concentrate in bodily compartments where it disrupts cellular processes and can result in detrimental health consequences. The concentration and impact of lead within follicular fluid has not been characterized and we used inductively coupled plasma mass spectroscopy (ICP-MS) to determine lead levels in blood and follicular fluid from nine patients undergoing in vitro fertilization (IVF) treatment. Lead levels within follicular fluid were found to be significantly higher in non-pregnant patients compared to pregnant patients suggesting that elevated concentrations of the environmental toxicant lead adversely affect female reproduction.