Comparison between human serum and Albuminar-20 (TM) supplement for in-vitro fertilization

Patient or fetal cord serum is commonly used as a protein supplement to culture media used in in-vitro fertilization (IVF). To eliminate the variability and possible hazards related to the use of human serum, a well-defined protein supplement, Albuminar-20 (Armour Pharmaceutical Cy) was evaluated as a substitute for serum. Prior to its application in the human, Earle's culture media supplemented with 0.5% (w/v) bovine serum albumin, 8% (v/v) decomplemented patient serum or 2.25% (v/v) Albuminar-20 were compared in a mouse bioassay. For the three different conditions, the percentages of blastocysts formed after 120 h in-vitro culture were respectively 91.2, 85.2 and 87.8% (NS). In the human IVF, a controlled comparison was performed from October to December 1988, between Earle's medium supplemented with patients' serum or Albuminar-20. When oocytes and spermatozoa were cultured in these two media, the fertilization rates were similar, 58.9% in human serum versus 59.4% in Albuminar-20. After further culture, the morphological quality of the cleaved embryos was better in the embryos cultured in Albuminar-20. The higher pregnancy rate in Albuminar-20 was correlated with the better morphological appearance of the embryos and their more advanced cleavage stage at the time of transfer. Therefore, Albuminar-20 can be considered as a suitable protein supplement in human IVF.     

Improvement of the culture conditions for the development of human preimplantation embryos

The culture of human preimplantation embryos from the 1-cell to the morula/blastocyst stage of development is not satisfactory at present. The success of various IVF laboratories ranges from 18 to 23%, therefore there is a requirement for improvement in the standard conditions used to culture the embryo. Using a limited number of 'spare' human embryos which were donated for research, in-vitro studies have been undertaken using various culture media. The results show that a significant improvement in viability is achieved using Ham's F-12 medium compared with other media presently used for culturing embryos.     

Medium-associated luteinization expressed as progesterone release in granulosa-luteal cells isolated from patients undergoing in-vitro fertilization.

The present study describes the effect of culture medium components on progesterone release from human granulosa-luteal cells isolated from patients undergoing in-vitro fertilization (IVF). Progesterone release was selectively measured as a central parameter of in-vitro luteinization, a process believed to decrease the success rate of IVF treatments. Ten different media of relevance to embryo culture were investigated for their effect on progesterone release in unstimulated granulosa cell cultures and in cultures stimulated with human chorionic gonadotrophin (HCG) (1 IU/ml) during 4 days in vitro. Culture media supplemented with human serum yielded the greatest secretion of progesterone. Supplementation with fetal calf serum caused an intermediate pattern of progesterone release. Substitution of serum with a synthetic replacement (Medi-CultR SSR 1 and 2), lacking hormones, cholesterol and growth factors, led to a minimal output of progesterone from granulosa-luteal cells. Complex media (RPMI 1640 and Ham's F10) generally caused a greater progesterone release than simple salt solution (EBSS). No effect of insulin was detected when added to serum-free media.     

Effect of the number of inseminated spermatozoa on subsequent human and mouse embryonic development in vitro.

It has been shown, in both human and mouse in-vitro fertilization (IVF), that an excess number of spermatozoa in the insemination medium leads to reduced fertilization rates. In this study, we evaluated human embryonic development after dividing the oocytes of each of 62 IVF attempts into two groups on the basis of insemination with two widely used concentrations (50,000 and 100,000 spermatozoa/ml). The embryonic growth was retarded in the group inseminated with 100,000 spermatozoa/ml: significantly fewer fast developing embryos (4-cell and 5- to 8-cell stages) were found (53.4% in the 100,000/ml group and 65.5% in the 50,000 group; P less than 0.05). In two experimental series, mouse embryonic development was evaluated in the presence of 0, 50,000, 100,000 and 500,000 spermatozoa per ml. In the first series, the spermatozoa were present during 5-20 h after insemination, while in the second series, the spermatozoa were present during the whole culture period of 120 h. The development of mouse embryos was impaired when 500,000/ml spermatozoa were present during the whole culture period. In contrast with human IVF results, the presence of up to 500,000 spermatozoa during the first 20 h after insemination did not have any significant detrimental effect on blastocyst formation in the mouse.     

Endocrinology: Immunoactive interleukin-1{beta} and tumour necrosis factor-{alpha} in thecal, stromal and granulosa cell cultures from normal and polycystic ovaries

Previous studies of follicular fluid from stimulated human ovarieshave shown detectable amounts of immunoactive interleukin-1(IL-1) and tumour necrosis factor- (TNF). Other reports haveshown the presence of IL-1 receptors, mRNA and antagonists forIL-1 in the human ovary. The aim of this study was to measureIL-1 and TNF concentrations in follicular fluid from unstimulatednormal or polycystic ovaries (PCO) as well as concentrationsin media conditioned by granulosa cells or thecal or stromaltissue. TNF concentrations were easily detected in follicularfluid (77 fmol/ml, range 20–95) and concentrations inPCO were similar to those in normal ovaries (70 versus 82 fmol/ml). TNF was virtually undetectable in all tissue culture media.IL-1 concentrations in all culture media were readily detectedbut showed no differences between different tissues or betweenPCO and normal ovaries. It was concluded that human ovariesreadily produce immunoactive IL-1 in culture but produce lessTNF despite detectable amounts of TNF in follicular fluid. Thereappears to be no difference between PCO and normal ovaries withrespect to IL-1 and TNF.     

Culture and selection of viable blastocysts: a feasible proposition for human IVF?

In human in-vitro fertilization (IVF) embryos are routinelytransferred to the uterus on day 2 or day 3 of development.Resultant implantation and pregnancy rates are disappointinglylow, with only 10% of embryos transferred leading to a livebirth. The ability to culture embryos to the blastocyst stageshould help to resolve this problem by synchronizing the embryoswith the female reproductive tract, and by identifying thoseembryos with little developmental potential. Co-culture hasoffered a possible means of producing blastocysts capable ofhigh implantation rates. However, recent developments in thefield of embryo physiology and metabolism have led to the formulationof new sequential serum-free culture media capable of supportingthe development of viable blastocysts in several mammalian species,including the human. It is therefore proposed that blastocysttransfer should be considered for routine use in human IVF.The high viability of blastocysts cultured in the appropriatesequential media means that fewer embryos are required for transferto achieve a pregnancy, culminating in fewer multiple births.Furthermore, the development of suitable non-invasive testsof embryo viability should further increase the overall successof human IVF by the ability to select before transfer thoseblastocysts most able to establish a pregnancy.     

Frozen storage of Ham's F10 medium for human in-vitro fertilization

A method of frozen storage of Ham's F10 medium was investigated that provides 'ready-to-use' culture medium for human in-vitro fertilization, without the necessity of readjusting and testing the medium after thawing. Ham's F10 medium, without bicarbonate, was adjusted to 245 mOsm/kg and stored in aliquots of 33 ml at -20 degrees C. Aliquots of 1 ml of a 7.5% (w/v) sodium bicarbonate solution were stored separately at the same temperature. The two components were mixed together after thawing. In the first test series, mouse embryos were cultured in media stored frozen for varying intervals between 2 weeks and 6 months and no difference in the rates of blastocyst formation was detected. Frozen-stored Ham's F10 medium was then used for human IVF in 256 cycles performed within a 16-month period in two different IVF centres. The pregnancy rates were evaluated and correlated with the duration of the frozen storage (between 1 week and 3 months) and compared to the outcome of 24 cases in which non-frozen medium was used. There was no significant difference in the pregnancy rates in the different groups (19% with non-frozen medium and between 21 and 33% with frozen-stored medium). Thus it was shown that there is no loss of quality of the frozen-stored media within the tested period of 3 months. The prolonged storage interval offers the possibility of extended quality tests and cross-tests between different IVF laboratories.     

A retrospective analysis of the in-vitro development of 'spare' human in-vitro fertilization preimplantation embryos using 'in-house' prepared medium and 'Medi-Cult' commercial medium

In-house prepared medium was used routinely in our in-vitrofertilization (IVF) facility prior to the introduction of thecommercial ‘Medi-Cult’ products. A comparative studyof the in-vitro development of embryos cultured in two [T6 andEarle's balanced salt solution (EBSS)] humaninactivated serum(HlS)-supplemented media from days 0 to 5 showed that 44.7%(46/103) of the embryos developed to the blastocyst stage inthe T6 medium compared with 22.3% (23/103) in EBSS. Followingthe introduction of the commercial Medi-Cult IVF M2 medium,which is used routinely to culture fertilized eggs from days0 to 2, new baseline data were required for the in-vitro developmentof ‘spare’ embryos from days 2 to 5. When Medi-CultM3 medium was used, 35.6% (37/104) of the ‘spare’day 2 embryos achieved the blastocyst stage. However, if morphologicallysimilar (four normal nucleated blastomeres with no fragmentation)day 2 embryos were selected, an increase in the blastocyst rateto 50.0% (33/66) was achieved. This compared favourably withthe 45.0% blastocyst rate (published in the Medi-Cult literature)for M2/M3 medium cultured human embryos. A small series of experimentswith T6 $ HIS medium and human serum albumin (HSA)- supplementedHam's F-10, MCDB 302 and M3 media was undertaken to identifya suitable medium which could be used for the culture of M2medium day 2 embryos. Results show that M2 medium cultured embryosplaced in Ham's F-10 medium supplemented with 10 mg/ml HSA gavean acceptable 37.8% (14/45) blastocyst rate. Therefore, thismedium could be substituted for M3 medium in an emergency. Atotal of 483 IVF embryos donated by patients, which were surplusto the therapeutic IVF programme, were used for these studiesover a period of 30 months. Late day 2 IVF spare embryos wereassigned an embryo score based on a high-power phase-contrastmicroscopic examination prior to being placed in culture. Theembryo score provides an effective in-vitro parameter with whichembryos from different patients can be compared. The cleavageand development of individual embryos were monitored on days2 to 5. In some cases, the continuing normal development andviability of the day 5 cultured embryo were assessed by monitoringthe hatching, attachment and outgrowth of the cavitated blastocyst.     

Dimeric inhibins and activin A in human follicular fluid and oocyte-cumulus culture medium.

The concentrations of inhibin A, inhibin B and activin A in follicular fluid and oocyte culture medium were analysed to investigate the production of these peptide hormones by ovarian granulosa cells and oocyte-cumulus complexes, as well as their potential as possible biochemical markers for oocyte quality and fertilizing capacity. Follicular fluids were collected from individual follicles during oocyte retrieval for in-vitro fertilization (IVF). Oocyte-cumulus culture media were collected after in-vitro insemination. The concentrations of dimeric inhibin A, inhibin B and activin A were measured using two-site enzyme-linked immunosorbent assays in the follicular fluid and matched oocyte culture medium. Hormone concentrations were compared with oocyte quality and fertilizing capacity. The concentration of inhibin A in follicular fluid increased while that of inhibin B decreased with increasing follicle size. Follicular fluid concentrations of inhibin A inhibin B and activin A were not significantly different in follicles with differing oocyte quality. Oocyte culture medium concentrations of activin A were significantly higher in morphologically good quality oocytes. There was no relationship between the concentrations of the three hormones and oocyte fertilizing capacity. This study confirms that follicular fluid concentrations of inhibin A may prove to be a marker of follicular growth and maturation. Higher concentrations of activin A produced by good quality oocyte-cumulus complexes suggest that activin A may play a role in oocyte maturation.     

IVF within microfluidic channels requires lower total numbers and lower concentrations of sperm

BACKGROUND: Microfluidic technology has been utilized in numerous biological applications specifically for miniaturization and simplification of laboratory techniques. We sought to apply microfluidic technology to murine IVF. METHODS: Microfluidic devices measuring 500 microm wide, 180 microm deep, and 2.25 cm in length were designed and fabricated using poly(dimethylsiloxane) (PDMS). Controls were standard centre-well culture dishes with 500 microl of media, half of which also contained PDMS as a material control. Denuded mouse oocytes were placed into microchannels or centre-well dish controls in groups of 10, then co-incubated overnight with epididymal mouse sperm at various concentrations. Fertilization was assessed and Fisher's exact test was used for statistical analysis (P < 0.05 significant). RESULTS: Fertilization rates between the two control groups (42%, no PDMS; 41%, with PDMS; not significant) were similar. Fertilization rates for denuded oocytes at standard mouse insemination sperm concentration (1 degrees 10(6) sperm/ml) was poorer in microchannels (12%) than controls (43%; P < 0.001). As insemination concentrations decreased, fertilization rates improved in microchannels with a plateau between 8 degrees 10(4) and 2 degrees 10(4) sperm/ml (4000-1000 total sperm). At these concentrations, combined fertilization rate for denuded oocytes was significantly higher in microchannels than centre-well dishes (27 versus 10%, respectively; P < 0.001), and was not significantly different from corresponding controls with a sperm concentration of 1 degrees 10(6) (37%; P = 0.06). CONCLUSIONS: Murine IVF can be conducted successfully within microfluidic channels. Lower total numbers and concentrations of sperm are required. Microfluidic devices may ultimately be useful in clinical IVF.     

Premature chromosome condensation as a sign of oocyte immaturity.

In this work we report the possibility that oocyte immaturity is associated with premature chromosome condensation (PCC) after in-vitro fertilization (IVF). Using a murine model, we have related PCC and endoreduplicated-like oocytes to oocyte immaturity as a basis for a prognosis in oocyte immaturity problems. The cytogenetic analysis was performed in 511 embryos obtained from immature oocytes that were directly fertilized in vitro and in 1363 embryos obtained from immature oocytes that were matured in vitro with different concentrations of human chorionic gonadotrophin (HCG) added to the culture medium. As a control we used 507 embryos obtained from freshly ovulated oocytes. PCC at the G1-phase-(G1-PCC) was observed only when immature oocytes were immediately fertilized in vitro (45.4%) and PCC at the S-phase (S-PCC) only when using in-vitro matured oocytes with the highest HCG concentration (3.3%). Neither G1-PCC nor S-PCC were found in the control group. Endoreduplicated-like oocytes appeared in a significant percentage (27.3%) only in the immature group. Immature oocytes yielded a low fertilization rate (16.6%) while in-vitro maturation seemed to confer a higher fertilization capacity compared to the control group (90.1% versus 78.2%). The possible correlation between PCC and oocyte immaturity provides new prospects in the determination of human IVF failures of unknown origin. Thus, when a problem of oocyte immaturity is diagnosed through the presence of PCC, a special programme of in vitro oocyte maturation, such as a longer preincubation time or addition of hormones to the media, would be recommended.     

Fertilization and early embryology: The applicability of the cumulative embryo score system for embryo selection and quality control in an in-vitro fertilization/embryo transfer programme

The cumulative embryo score system involves three aspects ofrelevance in pregnancy achievement during in-vitro fertilization(IVF) and embryo transfer: cleavage rates, morphological qualitiesand the number of embryos transferred. The scores of 602 IVF/embryotransfer trials were calculated and analysed to determine thesystem's relationship to pregnancy rate, pregnancy outcome andthe incidence of twin and triplet pregnancies. The system wasalso applied to cycles where endotoxins were either presentin or absent from culture medium, in order to evaluate its validityin quality control analyses. Pregnancy rates were found to increasefrom 4%, with scores between 1 and 10, to 35% in the 41–50group. The score of 20 was the criterion for separating patientsinto poor and good pregnancy prognosis groups (P = 0.00001).Biochemical abortions occurred more frequently with scores <20(P = 0.00978), but a similar relationship was not found in clinicalabortion rates (P = 0.62206). Birth rates below and above ascore of 20 (2.8 and 19.2%, respectively) differed significantly(P = 0.0005). The scores of twins overlapped extensively withthose of singleton births, but those of all triplets were >40. The system did not reflect a correlation between embryoquality and the presence of endotoxins in culture medium.     

Embryonic platelet-activating factor: an indicator of embryo viability

BACKGROUND: A definitive need exists to identify a biomarker of embryonic viability. Platelet-activating factor (PAF) production by human embryos is related to pregnancy potential. METHODS: Conditioned embryo culture media were obtained following conventional IVF on day 3, with PAF levels and pregnancy outcomes correlated. RESULTS: Overall pregnancy rate was 68% (17/25) with a mean of 84.1 (+/- 8.5) pmol/l/embryo PAF level. PAF levels ranged from a 216.4 pmol/l/embryo (pregnant) to a 3.7 pmol/l/embryo (not pregnant). There was a significant difference (P < 0.05) in PAF content between pregnant (92.1 +/- 9.5 pmol/l/embryo) and non-pregnant groups (52.5 +/- 16.6 pmol/l/embryo). Patients were categorized into three groups based upon PAF levels: low (< or= 5 pmol/l/embryo); medium (51-100 pmol/l/embryo) and high (>100 pmol/l/embryo). The low (60%) group had a significantly (P < 0.05) lower pregnancy rate than either the medium (85%) or high (89%) groups. A receiver-operator characteristic curve predicted a cut-off limit of 45 pmol/l/embryo for PAF content in human embryo conditioned culture media. CONCLUSIONS: The data demonstrate a correlation between PAF levels in human embryo conditioned culture media and pregnancy outcome. Additionally, as embryonic PAF levels increase so does the corresponding pregnancy rate. Therefore, PAF may be used as an indicator of embryo viability and for predicting pregnancy outcome.     

Formulation of a protein-free medium for human assisted reproduction

The optimal concentrations of individual amino acids, antioxidants, vitamins, osmolytes and energy sources were determined using a 1-cell Swiss outbred (SO) and or F(1) [(CBAxC57BL/6J)xSO] mouse assay in Earle's balanced salt solution containing bovine serum albumin. Based on the findings of these experiments, a number of media were formulated. Of these, the medium showing optimal embryo development and a significantly higher blastocyst hatching rate was investigated further. A protein-free medium (ART-7) was formulated and assessed using 1-, 2- and 4-cell SO mouse embryos. The generation of viable human embryos in the ART-7 series of media in micro- and ultra micro-droplet culture under oil with and without cumulus co-culture following intracytoplasmic sperm injection (ICSI) was investigated. The quality of sibling day 2 human embryos generated in the ART-7 media series was statistically comparable to or better than control embryos. The ART-7 medium was not toxic to human spermatozoa. Fertilization by conventional IVF and subsequent embryo development was not affected. A clinical trial of ICSI-derived embryos generated in the protein-free medium, with and without cumulus co-culture, has resulted in clinical pregnancies (10 of 20 transfers) of which two have proceeded to term, and the remaining patients are in various stages of pregnancy.     

淫羊藿苷对猪早期胚胎体外培养一氧化氮及丙二醛水平的影响

目的 研究淫羊藿苷(ICA)单体对猪体外受精早期胚胎体外发育过程中培养液内一氧化氮(NO)和丙二醛(MDA)生成量的影响,探讨其与猪胚胎体外发育效果的关系. 方法 以NCSU-23培养液为对照组,以添加0.6mg/L淫羊藿苷为实验组(ICA组),通过硝酸还原酶法和硫代巴比妥酸法测定猪体外受精卵体外培养0～48h、48～120h、120～168h培养液滴中NO和MDA水平. 结果 ICA组(342枚受精卵)48h卵裂率(74.84% vs 63.32%)、120h桑囊率(45.08% vs 34.10%)、168h囊胚率(23.38% vs 18.04%)均极显著高于对照组(349枚受精卵) (P<0.01).在猪体外受精卵体外培养0～168h整个过程中,培养液内NO、MDA水平总体均呈上升趋势,但在胚胎发育3个阶段中ICA组平均每枚胚胎NO生成量高于对照组,MDA生成量则低于对照组 (P> 0.05). 结论 ICA有利于促进猪体外受精早期胚胎的体外发育,可能与其在一定程度上提高NO水平,并同时抑制MDA的生成有关.     

Relaxin secretion by human granulosa cell culture is predictive of in-vitro fertilization-embryo transfer success

We have developed a cell culture system for human luteinizing granulosa cells which supports the timely and dynamic secretion of oestrogen, progesterone and relaxin in patterns that mimic serum concentrations of these hormones during the luteal phase of the menstrual cycle. There was a wide variation in the amount of relaxin secreted by the cultured cells for the 69 patients studied. As relaxin production was generally maximal by day 10 of culture, comparisons were made at this time point. It was observed that most of the conceptions occurred in patients with higher relaxin secretion in vitro. All cycles with relaxin > 800 pg/ml on day 10 had a term pregnancy while only 13% of cycles with relaxin < 200 pg/ml had term pregnancies. A limited number of cycles from donor/recipient cycles did not show similar results. Steroid concentrations were not predictive of conception. These results demonstrated that in-vitro production of relaxin is predictive of implantation success in in-vitro fertilization (IVF)-embryo transfer cycles. This supports the hypothesis that relaxin may be involved in implantation and that lowered relaxin concentrations may be a partial cause of poor pregnancy rates after IVF.     

Protein tyrosine phosphorylation, hyperactivation and progesterone-induced acrosome reaction are enhanced in IVF media: an effect that is not associated with an increase in protein kinase A activation

Sperm capacitation is a prerequisite for successful in vitro fertilization (IVF) and therefore a focus of sperm preparation in IVF laboratories. The technology of IVF is, therefore, potentially valuable in advancing our understanding of the molecular processes that occur during sperm capacitation. We have investigated sperm capacitation induced by a commercial IVF medium compared to that occurring in standard capacitating medium (CM) typically used in a nonclinical setting. Percoll-washed spermatozoa were resuspended in Cook Sydney IVF medium, Cook Sydney IVF sperm buffer, Earle's balanced salt medium (capacitating medium) or a modified Earle's balanced salt medium [non-capacitating medium (NCM)] for up to 120 min at 37 degrees C and, if applicable, in the presence of 5% CO2 in air. Sperm protein kinase A (PKA) activity, PKA-dependent serine/threonine phosphorylation, tyrosine phosphorylation, hyperactivation and progesterone-induced acrosome reaction were evaluated. IVF medium was shown to accelerate sperm capacitation (compared with capacitating medium) as determined by tyrosine phosphorylation, sperm hyperactivation and progesterone-induced acrosome reaction. This effect was not associated with enhanced activation of PKA or increased levels of serine/threonine phosphorylation. In contrast, IVF sperm buffer (used for sperm preparation) did not stimulate sperm capacitation when incubated for up to 90 min. We have shown that different capacitating media vary strikingly in their efficacy and that this difference reflects activation of a pathway other than the well-characterized activation of soluble adenylyl cyclase/cAMP/PKA.     

Why does hydrosalpinx reduce fertility? The importance of hydrosalpinx fluid

The debate on hydrosalpinx and impaired IVF outcome has mainly focused on the best treatment before IVF and on functional surgery as an alternative treatment. We would like to initiate a debate on the possible reasons why the outcome is impaired. We know that salpingectomy is effective in terms of improved birth rates after IVF, but we do not know exactly why. The main focus is on embryotoxic properties of the hydrosalpinx fluid, which include micro-organisms, endotoxins, cytokines, oxidative stress and lack of nutrients. The endometrial receptivity may be reduced as an effect of disturbed expression of the cytokine cascade, which is essential for implantation. The presence of excessive fluid in the uterine cavity may also be a mechanical hindrance to implantation. We believe that the hydrosalpinx fluid is of crucial importance, but the actual mechanism of action needs to be clarified.     

A new cell culture assy for quality control in IVF

A new, simple and sensitive bioassay for quality testing inIVF is described. This bioassay (Medi-Cult® Hybritest, GEALtd, Biotech Division, Hvidovre, Denmark) is based on the cultureof the rapidly growing mouse hybridoma cell line 1E6 in a definedserum-free culture medium. The use of serum-free conditionsgreatly increases the sensitivity to toxic substances, due tothe absence of binding proteins. The testing of known toxicagents showed that this assay disclosed cytotoxicity with ahigh sensitivity. The Hybritest thus provides a simple yet sensitiveand reproducible bioassay for quality control of culture media,water, chemical compounds and equipment in an IVF programme.Tests of different batches of culture media showed that mediafor IVF should be processed from powder and high-quality sterilewater. It is important not only to test the single componentsof the culture media, but also the final product in a sensitivetest system.