Fertilization and early embryology: Controlled comparison of Earle's balanced salt solution with Menezo B2 medium for human in-vitro fertilization performance

In order to optimize the in-vitro fertilization (IVF) procedureand to avoid the batch-to-batch fluctuations of in-house culturemedium, we investigated the use of a commercially availablemedium. In our human IVF programme, a controlled comparisonwas performed from July 1991 to February 1992 between in-houseEarle's medium and commercially available MénézoB₂ medium. The major difference between the two tested mediais the additional presence of amino acids and water-solublevitamins in Ménézo B₂ and the different originsof the protein source, i.e. bovine serum albumin (BSA) in MénézoB₂ and human serum albumin (HSA) in Earle‘s medium. Inthe first, auto-controlled study, sibling oocytes cultured inMénézo B₂ showed a significantly higher fertilizationrate than those cultured in Earle’s medium: 58.1±37.2% and 52.1±36.9% respectively. After further cultureof the fertilized oocytes, there was no difference in the morphologicalcharacteristics of the embryos cultured in the different media,although the rate of development was different. In MénézoB₂ medium, a higher proportion of the embryos had reached atleast the 4-cell stage at transfer. In the second, randomizedstudy, effects on the pregnancy and implantation rates of bothmedia were analysed. In the group of oocytes cultured in B₂,more excellent or good-quality embryos were available for transferat a more advanced stage of development. No significant differencein pregnancy rate was observed: 39.1% per transfer in Earle‘smedium versus 40.5% per transfer in Ménézo B₂.The implantation rate per embryo as well as the ongoing implantationrate per embryo did not differ between the two media: 21.0 and15.0% respectively for Earle’s medium and 20.5 and 14.3%respectively for Ménézo B₂ medium.     

A prospective randomized comparison of sequential versus monoculture systems for in-vitro human blastocyst development

BACKGROUND: Extending the period of in-vitro culture to the blastocyst stage may improve implantation rates in IVF treatment. Recognition of the dynamic nature of early embryo metabolism has led to the development of commercially available sequential culture systems. However, their improved efficacy over monoculture systems remains to be demonstrated in prospective studies. METHODS: Embryos obtained from 158 women undergoing IVF treatment were randomized by sealed envelopes to culture in one of three systems: (A) culture for 5 days in our own monoculture medium (Rotterdam medium); (B) culture for 3 days in Rotterdam medium followed by 2 days in fresh Rotterdam medium; (C) culture for 5 days using the commercially available G1/G2 sequential culture system. RESULTS: There were no significant differences in blastulation, implantation or pregnancy rates between the three tested culture systems. CONCLUSION: The employed monoculture system is as effective as the G1/G2 sequential system for the culture of blastocysts for IVF.     

Isolation of a histamine releasing factor from human embryo culture medium after in-vitro fertilization

In-vitro fertilization (IVF) of human oocytes in our laboratories gave a percentage pregnancy rate per transfer close to 20% during 1985. Embryos were grown until the two-four cell stage and then transferred to the maternal uterus. The media from these embryo cultures were collected and subjected to chromatography on heparin-Sepharose affinity columns. The bound protein fraction contained a factor capable of inducing histamine release from sensitized basophils. The effect of this embryo-derived histamine-releasing factor (EHRF) was to induce a maximum 56 +/- 7% release of the total histamine available. This value varied between 20 and 60%, resulting from 10-30 micrograms/ml of EHRF. Since the histamine release assay performed with basophils from non-atopic donors gave no positive results, we conclude that the release was not due to a cytotoxic mechanism. This was also supported by the absence of histamine release when the assay was performed at 0 degree C, or in the presence of 2 mM EDTA, suggesting that release was dependent on an immunological interaction between EHRF and some receptor on the basophils. The immunosuppressive role of histamine is well known, and a model involving EHRF and histamine is suggested here to explain the mechanism mounted by the embryo to escape maternal immune rejection.     

A comparison of three gonadotrophin-releasing hormone analogues in an in-vitro fertilization programme: a prospective randomized study

The use of gonadotrophin-releasing hormone analogues (GnRHa) has resulted in improved pregnancy rates in in-vitro fertilization (IVF) treatment cycles. Traditionally, short-acting analogues have been employed because of concerns over long-acting depot preparations causing profound suppression and luteal phase defects adversely affecting pregnancy and miscarriage rates. We randomized 60 IVF patients to receive a short-acting GnRHa, nafarelin or buserelin, or to receive a depot formulation, leuprorelin, all commenced in the early follicular phase and compared their effects on hormonal suppression and clinical outcome. We found that on day 15 of administration there was a significant difference in the suppression of oestradiol from initial concentrations, when patients on buserelin were compared with patients on nafarelin or leuprorelin (54 versus 72 and 65%; P < 0.05) and also in the number of patients satisfactorily suppressed, (80 versus 90 and 90%; P < 0.05), though there were no differences between the analogues by day 21. Similarly there was no difference in hormonal suppression during the stimulation phase or in implantation, pregnancy or miscarriage rates in comparing the three agonists. We conclude that with nafarelin and leuprorelin, stimulation with gonadotrophins may begin after 2 weeks of suppression and that long-acting GnRHa are as effective as short-acting analogues with no detrimental effects on the luteal phase.     

Evaluation of variations in pregnancy rates, implantation rates and early abortion rates within an in-vitro fertilization programme

Despite the application at this clinic of a standardized programmefor in-vitro fertilization of human oocytes over the last 27months, great variations in the rates of implantation, clinicalpregnancy and early abortion have been observed during certainperiods. A retrospective evaluation of these results showedthat these variations occurred in periods when various commerciallyavailable batches of Earle‘s medium (the medium was theonly variable changed during the 27 months) were used and thattwo sub-optimal batches of Earle’s medium from one ofthe sources used during one of three periods (period 2) wasmost likely to be responsible for sub-optimal embryo qualityand, consequently, for a halving of the pregnancy rate (30 versus15%) and of the implantation rate (11 versus 5%) and an increasein the early abortion rate (23 versus 50%), It is concludedthat the quality of the culture medium is of major importancefor the success of an IVF programme. The factor(s) in the mediumresponsible for the decrease in embryo quality has not beenidentified.     

A prospective, randomized comparison of ovulation induction using highly purified follicle-stimulating hormone alone and with recombinant human luteinizing hormone in in-vitro fertilization.

The commercial availability of highly purified, s.c. administered urinary follicle stimulating hormone (FSH) preparations for ovarian stimulation marked the beginning of a new era in the treatment of infertility. As these new formulations contain essentially no luteinizing hormone (LH), supplemental LH may be needed for optimal folliculogenesis. It was the aim of this pilot study to compare fertilization rates, embryo morphology, implantation rates and pregnancy outcomes prospectively in two age-matched patient groups: women who received highly purified FSH (FSH-HP) (n = 17), and women who received FSH-HP plus recombinant human LH (rhLH, n = 14) throughout ovarian stimulation. All patients received mid-luteal pituitary down-regulation with s.c. gonadotrophin-releasing hormone agonist (GnRHa) (leuprolide). Mean implantation rates were 26.9 and 11.9% in the FSH-HP only and FSH-HP + rhLH groups respectively. The mean clinical pregnancy/initiated cycle rate was 64.7 and 35.7% for the FSH-HP only and FSH-HP + rhLH patients respectively. FSH-HP patients and FSH-HP + rhLH patients achieved clinical pregnancy/transfer rates of 68.8 and 45.5% respectively. One patient in the FSH-HP + rhLH group had a spontaneous abortion; no pregnancy losses occurred in the FSH-HP only group. There were more cancellations for poor ovarian response among FSH-HP + rhLH patients (n = 3) than among FSH-HP patients (n = 1). The trend toward better pregnancy outcomes among patients who received FSH-HP without supplemental rhLH did not reach statistical significance. It is postulated that appropriate endogenous LH concentrations exist despite luteal GnRHa pituitary suppression, thereby obviating the need for supplemental LH administration.     

A prospective randomized trial of human chorionic gonadotrophin or dydrogesterone support following in-vitro fertilization and embryo transfer

A randomized, prospective blind study was carried out to investigate the need for luteal phase support in patients undergoing in-vitro fertilization (IVF). One-hundred-and-fifty-six patients undergoing IVF in cycles stimulated with human menopausal gonadotropin (HMG) and human chorionic gonadotrophin (HCG) stimulated IVF, were divided into three different groups for luteal phase treatment. Fifty-four patients received dydrogesterone three times daily (TID) beginning on the day of embryo transfer (ET). Fifty-one patients received HCG on days 3, 6 and 10 following ET. Fifty-one patients received placebo p.o. TID beginning on the day of ET. There was no difference between the groups in pregnancy rate, rate of spontaneous abortion, proportion of normally developing fetuses or rate of chemical pregnancy. The data indicate that supplementation of the luteal phase may not improve the success rates of IVF-ET cycles.     

Placental protein 14 in human in-vitro fertilization early pregnancies.

Placental protein 14 (PP14) and human chorionic gonadotrophin (HCG) were analysed in patients participating in an in-vitro fertilization-embryo transfer programme which did not include any kind of luteal support. Women with normal pregnancies, spontaneous abortions, ectopic pregnancies, biochemical pregnancies and non-pregnant women were compared. A combination of HCG and PP14 analyses distinguished between normal and abnormal implantation as early as 15 days after oocyte retrieval. The product of HCG (IU/l) and PP14 (micrograms/l) concentrations differed significantly between normal pregnancy, spontaneous abortion and ectopic pregnancy (P = 0.0248). It is concluded that both endometrial (PP14) and trophoblastic (HCG) markers, when used in combination, exhibit changes in abnormal implantation which may be clinically useful.     

Cytogenetic analysis of human oocytes and embryos in an in-vitro fertilization programme.

Oocytes (unfertilized and preovulatory) and embryos (normal and polypronuclear), which were donated to research by patients undergoing procedures of assisted reproductive treatment, were analysed for cytogenetic abnormalities. A total of 362 oocytes and embryos were analysed. The unfertilized oocytes with readable metaphases (53.4%) gave 25.2% chromosomal abnormality with diploidy being the main aberration observed. A high incidence of premature chromosome condensation (PCC) was observed and the incidence of PCC in oocytes exposed to colcemid was significantly higher (14/62, 22.6%) than in those not exposed to this treatment (3/41, 7.3%, P less than 0.05). When chromosomal anomalies and PCC in the unfertilized oocytes were correlated to various patient criteria such as stimulation regimen, number of human menopausal gonadotrophin ampoules, peak oestradiol levels, age of patient and number of previous attempts, none of the criteria tested had any significant relationship to the incidence of chromosomal abnormality. However a significant increase in the incidence of PCC was noted in the gonadotrophin-releasing hormone (GnRH) 'flare' group (6/15, 40.0%) compared to the GnRH 'down-regulation' group (11/88, 12.5%). The incidence of chromosomal abnormalities among preovulatory oocytes was 16.7% and diploidy was the only abnormality noted. For embryos arising from two-pronuclear oocytes, the chromosomal constitution related mainly to embryo quality. The rate of chromosomal abnormality for apparently good quality embryos was 23.5% and for poor or fragmented embryos 83.3%. The majority (77.3%) of the readable metaphase plates for polypronuclear 1-cell and cleaved embryos showed grossly abnormal chromosome complements but 19% of the cleaved embryos contained sets of normal diploid chromosomes.(ABSTRACT TRUNCATED AT 250 WORDS)     

A prospective controlled study of in-vitro fertilization, gamete intra-fallopian transfer and intrauterine insemination combined with superovulation.

The relative effectiveness of in-vitro fertilization (IVF), gamete intra-Fallopian transfer (GIFT) and intrauterine insemination (IUI) combined with superovulation in the treatment of infertility were compared in 151 couples undergoing a single cycle of treatment. Treatment was selected as appropriate (IVF for tubal disease, GIFT or IUI/superovulation for nontubal infertility) but possible bias due to non-randomization was overcome by all couples having had favourable fertilization in a previous cycle of IVF. Furthermore, in a preliminary study of initial IVF treatment in 265 couples from whom the study patients were drawn, implantation and pregnancy rates in the diagnostic groups were similar. In the definitive study comparing IVF, GIFT and IUI/superovulation, the pregnancy rate observed with GIFT was highest (40%) but this was not significantly higher than with IVF (28%) or IUI/superovulation (20%). However, the implantation rate per egg transferred by GIFT (21%) was significantly higher than the implantation rate per embryo transferred by IVF (11%). Although the pregnancy rates with GIFT were not statistically greater than with IVF, a significant advantage is likely to be observed in larger groups in view of the better implantation rate. The lower pregnancy rates with IUI superovulation are to be expected because of limited ovarian stimulation, they are nevertheless of comparative interest.     

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.     

Controlled comparison of conventional in-vitro fertilization and intracytoplasmic sperm injection in patients with asthenozoospermia.

A controlled comparison between conventional in-vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) has been carried out for patients with 相似文献   

Factors affecting pregnancy success of human in-vitro fertilization in unstimulated cycles

Successful pregnancies have recently been reported in cyclesof unstimulated in-vitro fertilization (IVF) which is a simplificationof the standard IVF approach utilizing ovarian stimulation.The purpose of this study was to analyse retrospectively theresults of the first 3 years of unstimulated IVF cycles at ourinstitution in order to identify factors which predispose thesecycles to success or failure. All patients (n= 57) underwentserial monitoring with transvaginal ultrasound and serum oestradioldeterminations. Human chorionic gonadotrophin (HCG) 10 000 IUwas administered when follicles were felt to be mature and aspirationundertaken (n= 98) 34–36 h later. Among nine patientsaged40 years, 13 aspirations resulted in nine embryo transfersand no pregnancies. In one completed cycle in this group, thepatient, who was 42 years old had a baseline follicle stimulatinghormone(FSH) concentration of 35.3 mIU/ml. The cycle progresseduneventfully and follicle aspiration yielded two oocytes andtwo morphologically normal embryos which, however, did not implant.In six patients <40 years with male factor, seven aspirationsyielded 18 oocytes of which 15 were inseminated and did notfertilize. One of the immature oocytes was allowed to maturein vitro and was fertilized and cryopreserved. Its transferin a subsequent cycle yielded a live birth. Among 78 cyclesin 42 patients aged <40 years without male factor, 63 resultedin embryo transfer with 14% clinical pregnancy rates per aspirationand 17% per embryo transfer. Pregnancy was associated with higheroestradiol concentrations at the time of HCG administrationand multiple embryos available for embryo transfer. There wasno effect on the pregnancy rate by the day of HCG administration,follicle diameter, number of cycles completed or number of oocytesobtained. We conclude that unstimulated IVF is a clinicallyviable alternative to stimulated cycles, that patients may beoffered up to three cycles without an appreciable decrease insuccess rates and that success rates which are highest amongwomen < 40 years old and in the absence of male factor maybe maximized by the attainment of maximal oestradiol coneetrationsprior to HCG administration     

Cryopreservation of human oocytes and fertilization by two techniques: in-vitro fertilization and intracytoplasmic sperm injection

Human oocyte cryopreservation results in poor survival and subsequentfertilization rates. It has been suggested that freeze-thaw-inducedchanges in the zona pellucida may impair sperm penetration orattachment. The aim of this study was to compare fertilizationand cleavage rates in cryopreserved oocytes inseminated by conventionalin-vitro fertilization (IVF) or intracytoplasmic sperm injection(ICSI). A total of 220 oocytes, obtained from volunteers whohad undergone ovarian stimulation, were cryopreserved usinga slow freeze-rapid thaw protocol with 1.5 M propanediol asthe cryoprotectant. Surviving oocytes (n= 74, 34.4%) were randomlyallocated for fertilization by conventional IVF (group 1) orICSI (group 2) using cryopreserved spermatozoa from a singledonor of proven fertility. Fertilization was achieved in five(13.5%) of the oocytes in group 1 and 17 (45.9%) in group 2(P < 0.005), with only one oocyte in group 1 exhibiting normalfertilization as opposed to 16 (43.2%) in group 2 (P < 0.001).Similarly, one oocyte fertilized by IVF cleaved, while all fertilizedwith ICSI cleaved (P < 0.001). We conclude that althoughthe survival of oocytes is poor following cryopreservation,fertilization and cleavage rates can be enhanced significantlyusing ICSI. These data also suggest that the method of cryopreservationused in this study affected the zona pellucida, such that normalsperm attachment or penetration was impaired.     

The frequency of chromosome anomalies in human preimplantation embryos after in-vitro fertilization

Previous studies have reported chromosome aberrations in humanpre-embryos after in-vitro fertilization (IVF). Although thereason for these abnormalities is not clear, there is evidencethat they can arise during gametogenesis, fertilization or cleavage.The present study has examined further the incidence of chromosomeabnormalities in human pre-embryos after IVF, using oocytesrecovered from normal volunteer women and from women undergoinginfertility treatment in an embryo-replacement programme. Chromosomepreparations were performed for 75 pre-embryos. Of these 35(47%) gave at least one metaphase in which analysis was possible.The overall incidence of abnormal pre-embryos was 40% (14/35).The absolute frequency of aberrations was 9% for trisomies,3% for polyploidies, 26% for structural anomalies and 3% forhypodiploidies. Five pre-embryos were found to be mosaics, threeof which had each one trisomic metaphase. In five of the pre-embryosmultiple anomalies were found. In 13 of the 14 abnormal pre-embryosthe aberrations were found in only one metaphase. The presentstudy demonstrates that trisomic mosaicism may not be a rareevent in human pre-embryos. Further evidence is provided thatmitotic non-disjunction is important for the production of aberrationsin human pre-embryos     

A prospective randomized study comparing the outcome of in-vitro fertilization and embryo transfer following culture of human embryos individually or in groups before embryo transfer on day 2.

A prospective randomized trial of in-vitro fertilization and embryo transfer was undertaken to investigate the reported beneficial effects of culturing preimplantation human embryos in groups, rather than individually. A total of 159 treatment cycles, in which the women were matched for age, basal gonadotrophin concentrations and number of previous attempts, were included in the study. Of these, 78 cycles were randomized to the 'individual culture' group, and 81 cycles were randomized to the 'group culture' group. The groups did not differ in terms of the median number of oocytes or embryos obtained per cycle. There was no statistically significant difference between the two groups in terms of treatment outcome, as assessed by pregnancies or clinical pregnancies.     

Costs in relation to effects of in-vitro fertilization.

A comparison has been made between in-vitro fertilization (IVF) and its most important alternative, tubal surgery. Medical results and financial costs have been taken into consideration. It is concluded that IVF must be regarded as a regular fertility treatment. The cost-effectiveness of IVF, in terms of the average treatment costs per full-term pregnancy, is approximately NLG 25,000, which is in line with that of tubal surgery. The deviation around the average is large, both for IVF and for tubal surgery.     

Effects of gonadotrophin-releasing hormone agonists on human ovarian steroid secretion in vivo and in vitro-results of a prospective, randomized in-vitro fertilization study

The aim of this prospective randomized study was to compare the effects of two gonadotrophin-releasing hormone (GnRH) agonists, buserelin and triptorelin, on human ovarian follicular steroidogenesis, oocyte fertilization and IVF treatment outcome. Ovulatory, healthy women undergoing IVF were treated either with human menopausal gonadotrophin (HMG) alone or with HMG and one of the two GnRH agonists. Serum and follicular fluid hormonal concentrations and cultures of luteinizing granulosa cells obtained during follicular aspiration were analysed. GnRH agonist treatment significantly affected steroidogenesis both in serum and follicular fluid. In follicular fluid, progesterone and oestradiol concentrations were significantly elevated while testosterone concentrations were significantly lower in the triptorelin group. The ratios of testosterone/progesterone, oestradiol/progesterone but not oestradiol/testosterone concentrations were significantly affected by GnRH agonist administration. Similarly, the steroidogenic activity of luteinizing granulosa cells in vitro was significantly decreased in women treated with GnRH agonists. Women treated with GnRH agonists had significantly more fertilized oocytes and cleaving embryos. The results indicate a marked effect of GnRH agonists on the pattern of ovarian follicular steroidogenesis that cannot be explained solely by changes in gonadotrophin concentrations.     

Quality control in an in-vitro fertilization laboratory: use of human sperm survival studies

A bioassay procedure is described for quality control testingof various disposable items used in routine IVF procedures.This biosassay is performed over 4 days and uses the survivalof human sperm in vitro at room temperature to assess whichproducts are suitable for use. New products were tested forcytotoxicity using a general screening method and subsequenthatches of every suitable item tested to detect interbatch variation.Products were considered suitable or unsuitable for use dependingupon a calculated sperm survival index. Two main types of productwere found to be cytotoxic, namely certain brands of syringeand surgical gloves, the common feature of both being the presenceof rubber components. The bioassay was also used to investigatefurther the cytotoxic effect of the powdered and starch-freesurgical gloves. The cytotoxic substances from both types ofsurgical glove were readily transferred to an embryo replacementcatheter by touch, and washing of the gloves reduced this effectonly moderately. The bioassay has proved inexpensive and convenientbut more importantly it has been invaluable for detecting potentialsources of cytotoxicity before they are introduced into a standardIVF protocol.     

Relationship between intrafollicular levels of prolactin and sex steroids and in-vitro fertilization of human oocytes

In this study the importance of Intrafollicular prolactin (PRL)and sex steroid levels, as indices of the potential invitrofertilization and development of human oocytes, was investigated.Oocytes were recovered from 20 normal female volunteers treatedwith a fixed schedule including norethisterone and clomiphene.The embryos which developed after in-vitro fertilization weregraded on a scale of 0 to 10 using morphological criteria. IntrafollicularPRL, oestradiol, progesterone and androstenedione levels didnot differ significantly between follicles containing oocyteswhich fertilized and those which did not fertilize in vitro.Nor did these hormones differ significantly between folliclesgiving rise to embryos with a bad score (0–5) and thosewith a good score (6–10). No significant correlation wasfound between serum and follicular fluid PRL levels. It is suggestedthat fertiliza tion and developmental potential of human oocytesin vitro cannot be predicted by the intrafollicular levels ofPRL and sex steroids at the time of laparoscopic egg recovery.