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.     

The questionable use of albumin for the prevention of ovarian hyperstimulation syndrome in an IVF programme: a randomized placebo-controlled trial

BACKGROUND: The role of intravenous (IV) albumin administration in the prevention of ovarian hyperstimulation syndrome (OHSS) and in the improvement of IVF conception outcomes was evaluated in a prospective, randomized, placebo-controlled double blind study. METHODS: Ninety-eight women were enrolled in the study and were consecutively assigned to either a treatment group or a control group. Eleven patients were lost to follow-up after assignment. Of the remaining 87 women, 46 received albumin on the day of oocyte retrieval, and 41 received 0.9% sodium chloride solution as a placebo control. Outcome measures included OHSS incidence rates and pregnancy rates in the two trial groups. RESULTS: Four of the 46 patients in the study group developed severe OHSS and six developed moderate OHSS. In the control group, one of the 41 developed severe OHSS and five developed moderate OHSS. The difference in OHSS incidence rates between the two groups was not statistically significant [relative risk (RR) = 1.49, 95% CI = 0.59-3.73]. Fourteen patients (30%) in the intervention group conceived, compared with 16 patients (39%) in the control group. The difference in conception rates between the two groups was not statistically significant (RR = 0.78, 95% CI = 0.44-1.39). CONCLUSIONS: Albumin appears to have no positive effect on OHSS or conception rates, while its use carries the risk of undesirable side effects, including exacerbation of ascites in OHSS, nausea, vomiting, febrile reaction, allergic reaction, anaphylactic shock and risk of virus and prion transmission. We suggest that this form of treatment should not be included in the prevention of OHSS.     

Similar delivery rates in a selected group of patients, for day 2 and day 5 embryos both cultured in sequential medium: a randomized study

BACKGROUND: The existence of a real benefit of blastocyst transfer is still a matter of debate. The aim of this study was to compare, in a prospective randomized trial, the outcome of day 2 and day 5 transfer of human embryos cultured in an 'in-house' sequential medium. METHODS: A total of 193 cycles from 171 patients with less than four previous IVF cycles, <39 years old and with four or more zygotes on day 1, were randomly allocated to day 2 (94 cycles) or day 5 (99 cycles) transfer. Zygotes were kept in fertilization medium until 18 h post-fertilization and then placed in a 'glucose-free' cleavage medium. Embryos allocated for day 5 transfer were placed in a blastocyst medium 66 h post-fertilization. Two or three embryos were replaced according to the morphology. RESULTS: A mean (+/- SEM) number of 2.1 +/- 0.4 and 1.9 +/- 0.3 embryos were replaced on day 2 and day 5 (P < 0.001) respectively. Delivery rates per transfer were 44.1 and 37.1% [P = not significant (NS)], implantation rates were 31.4 and 29.4% (NS) and multiple delivery rates 22 and 36% (NS) for day 2 and day 5 groups respectively. Ten patients (10.1%) had no blastocysts available for transfer. CONCLUSIONS: No clear benefits were observed using blastocyst transfer for patients aged <39 years who had had less than four previous IVF cycle attempts.     

Maternal serum HCG is higher in the presence of a female fetus as early as week 3 post-fertilization

BACKGROUND: Maternal serum HCG (MSHCG) is higher when the fetus is a female than when it is male. This has been demonstrated in the second and third trimesters of pregnancy, and recently at 10-14 weeks gestation. In this study we assessed whether this gender-related difference can be detected as early as week 3 post-fertilization. METHODS: The IVF setting was chosen because it provides precise dating of gestational age and early sonography for the number of gestational sacs. The study included 347 IVF cycles from 335 patients. Only pregnancies with a single implanted embryo that resulted in a single live birth of known gender were included. MSHCG was measured on days 14-20 post-fertilization, and levels were expressed as gestational age-corrected multiples of the median (MoMs). The log10 MSHCG MoMs were compared according to fetal gender. RESULTS: MSHCG levels were significantly higher (18.5%) in week 3 post-fertilization in the presence of a female fetus (P < 0.0002). CONCLUSION: Because a fetal gender-related difference in MSHCG can be demonstrated as early as week 3 post-fertilization, the difference may be attributed to placental factors and not to the effects of the fetal hypothalamic-hypophyseal-gonadal axis.     

Fertilization and early embryology: Absence of heat treatment of serum for culture medium supplementation does not adversely affect the outcome of in-vitro fertilization

This study was carried out to determine if not heat-treatingserum prior to use for medium supplementation adversely affectedin-vitro fertilization (IVF) of human oocytes. Morphologicallymature human oocytes derived from 135 patients undergoing IVFtreatment were studied. A total of 504 oocytes were incubated,inseminated and the resulting pronuclear oocytes cultured furtherin Earle's balanced salt solution (EBSS) supplemented with 10%non-heat-treated serum. Comparisons of fertilization rate andembryonic development were made between these and 687 controloocytes derived from the same patients but incubated, inseminatedand resulting pronuclear oocytes cultured further in EBSS supplementedwith 10% heat-treated serum. The fertilization rate of 74.4%(375/504) of oocytes handled in serum-supplemented medium thathad not been heat-treated was significantly better than therate of 67.7% (465/687) for controls (P< 0.0125). The proportionof pronucleate oocytes that cleaved was also significantly betterin the non-heat-treated serum group: 270/300 (90%) versus 307/375(81.8%) (P < 0.0025). There was no significant differencein the proportion of embryos with four or more cells at thetime of embryo transfer. The results show that the absence ofheat treatment of serum used to supplement culture medium hasno adverse effect on the fertilization rate and short-term embryodevelopment in vitro; hence we suggest that serum heat treatmentis an unnecessary procedure and could be abandoned.     

Development of embryos from natural cycle in-vitro fertilization: impact of medium type and female infertility factors

Single embryos derived from natural cycle in-vitro fertilization(IVF) were graded during the pre-transfer culture period usingmorphological criteria. Most embryos developed well in culturewith 96% showing continuing division and 68% showing good morphologicalappearance, although embryo quality tended to decline with anincreased incidence of fragmentation and uneven cleavage asdivision proceeded. Both the pregnancy rate and the distributionof embryo grades were similar among four different culture mediaused, suggesting that choice of medium had little impact onoutcome. In contrast, there were marked differences in pregnancyrate according to the type of infertility, which was not reflectedin a decrease in embryo quality. However, although embryos frompatients with tubal infertility implanted and formed viablepregnancies irrespective of morphological appearance, only ‘good’quality embryos from patients with non-tubal (or ‘unexplained’)infertility were able to implant. Thus the appearance of theembryo derived from natural cycle IVF in women with unexplainedinfertility may be of clinical relevance.     

Leukaemia inhibitory factor significantly enhances the blastocyst formation rates of human embryos cultured in serum-free medium

The aim of this study was to investigate the effect of leukaemiainhibitory factor (LIF) on human blastocyst formation ratesin vitro. To do this, it was first necessary to devise a complexserum-free medium (CSFM) in which to test its activity. Blastocystformation rates in CSFM microdrops (18.4%) showed no differenceto those obtained previously for embryos cultured in 1 ml T6medium containing 10% serum (25%). The majority of blastocystswere of optimal blastocyst grade (BG3). The percentage of BG1/BG2blastocysts was decreased in CSFM microdrops (10.2%) comparedto that observed in T6 medium (24.8%). Addition of 1000 IU/mlLIF to CSFM microdrops increased the blastocyst formation ratefrom 18.4 to 43.6% (P < 0.025) and increased the percentageof BG1/BG2 blastocysts (33%; P 0.025) to levels comparablewith those observed in T6 medium. Thus LIF significantly increasedthe quality and number of human blastocysts formed in CSFM medium,increasing the potential for blastocyst transfer in human in-vitrofertilization (IVF)     

The use of recombinant human LH (lutropin alfa) in the late stimulation phase of assisted reproduction cycles: a double-blind, randomized, prospective study

BACKGROUND: The effect of recombinant human LH (r-hLH; lutropin alfa) in women undergoing controlled ovarian stimulation with recombinant human FSH (r-hFSH) prior to IVF was investigated. METHODS: After down-regulation with the GnRH agonist, buserelin, 114 normo-ovulatory women (aged 18-37 years) received r-hFSH alone until the lead follicle reached a diameter of 14 mm. Patients were then randomized in a double-blind fashion to receive r-hFSH in addition to r-hLH, 75 IU s.c., or placebo daily for a maximum of 10 days prior to oocyte retrieval and IVF. The primary end-point was the number of metaphase II oocytes. RESULTS: There were no significant differences between treatment groups for the primary end-point. Serum estradiol concentrations on the day of HCG administration were significantly higher in the group receiving r-hLH plus r-hFSH than in the group receiving r-hFSH alone (P = 0.0001), but there were no significant differences between the groups in dose and duration of r-hFSH treatment required, oocyte maturation, fertilization rate, pregnancy rate and live birth rate. CONCLUSION: In this patient population, the addition of r-hLH during the late follicular phase of a long GnRH agonist and r-hFSH stimulation cycle provides no further benefit in terms of oocyte maturation or other end-points.     

The use of fibrin sealant for embryo transfer: development and clinical studies.

Many embryo transfers after in-vitro fertilization may fail because of expulsion of the embryos from the uterus. Approximately 5-8% of pregnancies resulting from embryo transfer are ectopic. The aim of our study was to find a technique to avoid ectopic pregnancies and to improve the pregnancy rate. We used a two-component fibrin sealant which also contains a fibrinolysis inhibitor (aprotinin) at various concentrations. After gaining experience with mouse embryos, the sealant was used in human embryo transfer with great success. The results of a pilot study encouraged us to perform a prospective randomized study on 546 patients (270 with fibrin sealant, 276 conventional embryo transfers). There were 47 (17.0%) orthotopic pregnancies and 6 (2.2%) ectopic pregnancies in the control group, whereas there were 51 (18.9%) intrauterine and no ectopic pregnancies in the treatment group. The difference in ectopic pregnancies was statistically significant (P less than 0.05). With regard to the aprotinin concentration, there was a trend towards better results with 100-150 kIU (28.5% clinical pregnancies) in comparison to 250-300 kIU (19.2%) or no aprotinin (20.4%) (not significant). Further improvements of the technique may raise the pregnancy rate when fibrin sealant is used. As shown in our prospective randomized study, ectopic pregnancies may be completely avoided.     

The use of fluorescent in-situ hybridization (FISH) for the analysis of in-vitro fertilization embryos: a diagnostic tool for the infertile couple

We use triple colour fluorescent in-situ hybridization (FISH)to sex human embryos for preimplantation diagnosis of X-linkeddisease, to analyse chromosome numbers in embryos donated forresearch purposes and as a diagnostic tool for patients undergoinginfertility treatment, especially in cases where abnormal embryodevelopment occurs. We have reported on the use of FISH in acase where all embryos showed accelerated cleavage. Here wereport on the use of triple colour FISH in a case where fiveout of seven oocytes were multi-nucleated when examined forpronuclei. The embryos were spread whole using HCI/Tween 20and triple colour FISH performed with probes for chromosomesX, Y and 1 in a 2 h procedure. Two embryos were normal for theprobes used, and three showed abnormalities, including one 4-cellembryo where all nuclei were X, X, X, Y, 1, 1, 1, 1. FISH indicatedthat fertilization had occurred, but that the majority of embryoswere abnormal confirming that such embryos should not be consideredfor transfer. In these cases, or where there is recurrent in-vitrofertilization failure or spontaneous abortions, embryos in futurecycles can be examined using FISH to ascertain the level ofchromosome abnormality which may aid future infertility treatment.     

A prospective randomized comparison of intramuscular or intravaginal natural progesterone as a luteal phase and early pregnancy supplement.

A luteal phase defect has been demonstrated in cycles stimulated using a protocol including a gonadotrophin releasing hormone agonist (GnRHa). We have conducted a randomized prospective study of luteal and early pregnancy supplementation in 262 women selected for in-vitro fertilization (IVF), gamete intra-Fallopian transfer (GIFT) or zygote intra-Fallopian transfer (ZIFT). Either intramuscular progesterone in oil (50 mg/day) or intravaginal micronized progesterone (600 mg/day) was used as luteal supplement. In association with oestradiol valerate, progesterone administration was initiated from the day before oocyte retrieval until the 12th week of pregnancy. The implantation rate just failed to reach statistical significance (P = 0.07) in favour of the group receiving intravaginal progesterone. In the latter group, we observed a higher clinical pregnancy rate (33.6 versus 26.7%, not significant). Despite lower plasma progesterone levels, a lower first trimester abortion rate (P less than 0.05) was found in the intravaginally treated group. Intravaginal micronized progesterone was well tolerated by all patients and appeared more effective than intramuscular progesterone in improving the implantation rate, and in decreasing the incidence of abortions in stimulated cycles including GnRHa.     

The incidence of major clinical complications in a Dutch transport IVF programme

Four different major clinical complications were identifiedin a retrospective analysis of 2495 in-vitro fertilization (IVF)cycles resulting in oocyte retrieval. The severe form of ovarianhyperstimulation syndrome (OHSS) occurred in 18 patients, givinga prevalence for this complication of 0.7%. Seven (39%) of these18 patients had previously been diagnosed as having polycysticovaries. Eleven patients were admitted with moderate OHSS. Adnexaltorsion was diagnosed in two patients. Ovariectomy was considerednecessary in both cases. Complications of the transvaginal procedureoccurred in seven cases (0.3%): one patient had an acute appendicitiswith puncture holes in the appendix, six patients were admittedshortly after oocyte retrieval with a pelvic inflammatory disease.Of the 624 pregnancies obtained, 13 were ectopic, giving anectopic pregnancy rate of 2.1%. It is concluded that seriousclinical complications of IVF treatment are rare. However, patientsshould be counselled for the occurrence of serious procedure-relatedcomplications before entering an IVF programme.     

The use of intrauterine insemination in Australia and New Zealand

BACKGROUND: There is good evidence in the literature in favour of intrauterine insemination (IUI) as the most cost-effective treatment for unexplained and moderate male factor subfertility. However there is no published data on whether this evidence is being translated into clinical practice. METHODS: We identified fertility centres within Australia and New Zealand registered with the Reproductive Technology Accreditation Committee of the Fertility Society of Australasia. Thirty-seven of these units were then sent a postal survey to establish current clinical practice. RESULTS: Nearly a third of centres promote IVF as first-line treatment even in the presence of patent tubes and normal semen while, when semen parameters are reduced, IUI is rarely considered. One in five (20%) units remain unconvinced of the cost-effectiveness of IUI. When IUI is used, it is virtually always combined with ovarian stimulation with marginally more units using clomiphene citrate than gonadotrophins. CONCLUSIONS: Although it may take relatively more treatment cycles to achieve pregnancy, there are considerable advantages to the patient in terms of risk/benefit ratio and financial cost associated with IUI compared with IVF. In the current climate of evidence-based medicine, as clinicians we are obliged to translate this into our practice. It appears from our survey that in many units this is not happening.     

Chemical composition and protein source in the capacitation medium significantly affect the ability of human spermatozoa to undergo follicular fluid induced acrosome reaction

We studied the effect of media composition on sperm capacitation,using Biggers—Whitten—Whittingham (BWW) medium,Ham's-F10 and a modified Tyrode's medium (HSM) supplementedwith bovine serum albumin (BSA) or fetal cord serum (FCS). Weevaluated the effect of chemical environment and protein supplementationon the sperm motion parameters of curvilinear velocity and linearity,and on the ability of incubated spermatozoa to undergo follicularfluid induced acrosome reaction. Neither chemical compositionnor protein supplementation of capacitation media greatly affectedmotion parameters after 2 h incubation. Furthermore, chemicalcomposition had only a small effect on the ability of spermatozoato undergo the acrosome reaction upon exposure to follicularfluid. A higher proportion of spermatozoa underwent acrosomereaction after incubation in HSM (8% control (C); 28% follicularfluid) than in BWW (8% C, 17% follicular fluid) or Ham‘sF-10 (6% C, 19% follicular fluid). By contrast, protein sourceproved critical in determining acrosome reaction inducibility.Spermatozoa incubated in BSA-supplemented media showed a 4-foldincrease in acrosomal discharge when exposed to follicular fluid(6% C, 22% follicular fluid) compared to controls while spermatozoaincubated in FCS were unable to undergo acrosome reaction (6%C, 6% follicular fluid). Simultaneous addition of FCS to BSAcapacitation medium blocked acrosome reaction inducibility andthe late addition of BSA, after sperm incubation in FCS, didnot facilitate acrosome reaction. We propose that an inhibitorof sperm capacitation is present in FCS and therefore, the selectionof optimum incubation conditions for spermatozoa may be of criticalimportance when evaluating or treating infertile patients.     

IVF of mouse ova in a simplex optimized medium supplemented with amino acids

The addition of amino acids to a modified simplex optimized medium (mKSOM) did not increase the percentage of blastocysts that develop from CF1 mouse ova fertilized in vitro. In contrast, the percentage of blastocysts that began to hatch and the number of cells in these blastocysts, particularly in the inner cell mass, was increased. The added amino acids also supported the development of a more organized extracellular matrix in the same blastocysts. The results suggest that zygotes produced in amino acid-supplemented mKSOM have a greater developmental potential, perhaps developing at a faster rate, than zygotes produced in mKSOM. This enhanced developmental potential may be caused by the alleviation of osmotic stress on the ova and zygotes by the amino acids that are osmolytes. The fertilization of human ova in vitro may benefit from the inclusion of free amino acids in the fertilizing medium. The availability of a medium that can be used to support both IVF and preimplantation development in the mouse is likely to benefit the recovery of mouse strains from cryopreserved spermatozoa.     

Interleukin-1 receptor antagonist gene (IL-1RN) polymorphism is a predictive factor of clinical pregnancy after IVF

BACKGROUND: Only 25% of IVF transfer cycles lead to a clinical pregnancy, calling for continued technical progress but also more in depth analysis of patients' individual characteristics. The interleukin-1 (IL-1) system and matrix metalloproteinases (MMPs) are strongly implicated in embryo implantation. The genes coding for IL-1Ra (gene symbol IL-1RN), IL-1beta, MMP2 and MMP9 bear functional polymorphisms. We analysed the maternal genetic profile at these polymorphic sites in IVF patients, to determine possible correlations with IVF outcome. METHODS: One hundred and sixty women undergoing an IVF cycle were enrolled and a buccal smear was obtained. The presence of IL-1RN variable number of tandem repeats and IL-1B + 3953, MMP2-1306 and MMP9-1562 single nucleotide substitutions were determined. Patients were divided into pregnancy failures (119), biochemical pregnancies (8) and clinical pregnancies (33). RESULTS: There was a 40% decrease in IL-1RN*2 allele frequency (P = 0.024) and a 45% decrease in IL-1RN*2 carrier status in the clinical pregnancy group as compared to the pregnancy failure group (P = 0.017). This decrease was still statistically significant after a multivariate logistic regression analysis. The likelihood of a clinical pregnancy was decreased accordingly in IL-1RN*2 carriers: odds ratio = 0.349, 95% confidence interval = 0.2-0.8, P = 0.017. The IL-1B, MMP2 and MMP9 polymorphisms showed no correlation with IVF outcome. CONCLUSIONS: IL-1RN*2 allele carriage is associated with a poor prognosis of achieving a pregnancy after 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.     

Quality control in the IVF laboratory: in-vitro and in-vivo development of mouse embryos is unaffected by the quality of water used in culture media

We have examined the effect of media made with tap water or with various purified waters on the fertilization of mouse oocytes, their development to blastocysts, their rate of hatching in vitro and their survival after transfer to recipients. Zona-intact and zona-free embryos, as well as cell clusters from 8-cell stage embryos, were also used. The macromolecular composition of the media was varied. We were unable to find any adverse effect of tap water under any condition examined. The implications of these findings for quality control in IVF units are discussed.     

Embryo scoring as a prognostic tool in IVF treatment

A semi-quantitative and non-invasive method for scoring embryosobtained after in-vitro fertilization (IVF) has been defined,aiming at selection of embryos before transfer and at prognosticevaluation of IVF trials. Grading of embryos observed on theinverted micrclscope was essentially based on the amount ofanucleate fragments expelled during early cteavage and on developmentalspeed. Embryos endowed with a high score were more often associatedwith pregnancy and in particular with the occurrence of maplepregnancy. No difference was observed between scores attributedto embryos related to ongoing, aborted or chankal pmgnades.Average embryonic scores corrrsponding to double and tripletransfers differed significantly in failures as well as pregnancies.The better quality of embryos replaced in triple transfers wasalso apparent from the significantly higher implantation rateper embryo observed in this group. From our results, five criteriaincluding clinical data and embryonic scores can be derivedfor defining a high risk of multiple pregnancy prior to transfer.It might be warranted to replace only two embryos when theseconditions are fulfilled.     

The use of a short regimen of buserelin, a gonadotrophin-releasing hormone agonist, and human menopausal gonadotrophin in assisted conception cycles

The outcome of in-vitro fertilization treatment using buserelin, an agonist of luteinizing hormone releasing hormone, given in a short stimulation regimen with human menopausal gonadotrophin (HMG), was compared with a conventional regimen including clomiphene citrate (CC). A total of 94 infertile women underwent cycles of treatment with intranasal buserelin, 500 micrograms daily from the first day of menstruation and also HMG daily from the third day. The same patients had previously undergone unsuccessful treatment cycles with CC and HMG. Overall, addition of buserelin resulted in fewer cycles being abandoned (10 versus 34%) and none of the patients ovulated prior to collection. The mean total dose of HMG required was increased by 74% in buserelin cycles. Significantly more oocytes were collected with buserelin treatment (mean 5.9 versus 4.4, P less than 0.01) and, thus, significantly more embryos were transferred (mean 2.3 versus 1.2, P less than 0.0001) although fertilization and cleavage rates were unchanged. Fifteen pregnancies were achieved, giving a clinical pregnancy rate of 22% per embryo transfer. These pregnancies resulted in 16 live births (7 singletons, 3 twins, 1 triplets). Four pregnancies failed before 14 weeks gestation. We conclude, therefore, that the substitution of buserelin for CC for ovarian stimulation in poor responders results in an improved outcome, both in terms of the number of oocytes collected and the pregnancy rate per treatment cycle.