Fertilization and early embryology: Nuclei number in human embryos co-cultured with human ampullary cells

A study was undertaken to evaluate the effect on nuclei numberin human embryos cultured in vitro with primary cell lines ofhuman Fallopian tube epithelium. The development of 203 surplushuman embryos, cultured in standard culture medium (Earle'sbalanced salt solution+15% A5) with or without ampullary cells,was observed from day 2 to day 5.5 post-insemination. The expandedblastocysts in both culture conditions were analysed for nucleinumbers per blastocyst. Embryos transferred to co-culture atthe 2-cell stage had an average of 120.7 nuclei per blastocyst,which was significantly higher than the average of 62.9 nucleiper blastocyst (P=0.023) for the embryos transferred to co-cultureat the 4-cell stage. The embryos cultured in the control mediumhad an average of 42.1 nuclei per blastocyst, which was significantlyless than co cultured embryos (P=0.04). Severely fragmentedembryos (grades 3 and 4) did not show recovery in co-culture.Our results show that when human embryos are transferred toco-cultures before the 4-cell stage, the blastulation rate andthe cell number per embryo increase significantly compared tothe embryos cultured in standard culture medium. The possibleeffect of co-culture on embryonic gene expression is discussed.     

Human preimplantation development in vitro is not adversely affected by biopsy at the 8-cell stage

Normally fertilized human embryos biopsied 3 days after in-vitro fertilization (IVF) have been examined for effects on viability and development in vitro after removal of one or two cells at the 8-cell stage (1/8 and 2/8) from each embryo. A high proportion of 7/8 and 6/8 biopsied and unmanipulated embryos developed to the blastocyst stage between days 5 and 6 (79, 71 and 59%, respectively), and many biopsied embryos (56%) hatched from the zona pellucida in vitro. The viability of biopsied embryos which developed to the blastocyst stage was assessed by daily non-invasive measurement of the uptake of two energy substrates, glucose and pyruvate. Uptake of both substrates was generally lower in 7/8 and 6/8 biopsied embryos but only in proportion to the reduced cellular mass. The total cell number and the numbers of both trophectoderm (TE) and inner cell mass (ICM) cells in biopsied embryos at the blastocyst stage, counted by differential labelling of their nuclei, were also reduced in proportion but the ratio of ICM to TE cells was maintained in both 7/8 and 6/8 biopsied embryos. We conclude that removal of one or two cells at the 8-cell stage, while reducing the cellular mass, does not adversely affect the preimplantation/development of biopsied embryos in vitro and suggest that this approach could be used for preimplantation diagnosis of genetic defects.     

The sperm centriole: its inheritance, replication and perpetuation in early human embryos

The inheritance, replication and perpetuation of the sperm centriolein the early human embryo are reported. Both normal monospermicand abnormal dispermic embryos (n = 127) were examined by transmissionelectron microscopy. Centrioles were traced from fertilizationto the hatching blastocyst stage. The sperm proximal centrioleis introduced into the oocyte at fertilization and remains attachedto the expanding spermhead during sperm nuclear decondensation,as it forms the male pronucleus. A sperm aster is initiallyformed after the centriole duplicates at the pronuclear stage.At syngamy, centrioles occupy a pivotal position on oppositespindle poles, when the first mitotic figure is formed. Bipolarspindles were found in the majority of embryos, while tripolarspindles were seen in four dispermic embryos at syngamy. Twosingle centrioles were detected at two poles of two tripolarspindles, while two additional centrioles were located on thesides of a bipolar spindle of a dispermic embryo. Sperm tailswere detected near spindle poles at syngamy and in later embryos.Typical centrioles showing the characteristic pin-wheel organizationof nine triplets of microtubules were evident During centriolarreplication, the daughter centriole grows laterally from theparent and gradually acquires pericentriolar material (PCM).The two centrioles are surrounded by a halo of electron-densePCM, which nucleates microtubules, thus making it a typicalcentrosome. The usual alignment of diplosomes at right anglesto each other was maintained. Centrioles were detected at allstages of embryonic cleavage from the 1-cell through 8-cellstages, right up to the hatching blastocyst stage. They wereclosely associated with nuclei at interphase, when they wereoften replicating, and were prominently located at spindle polesduring the first four cell cycles. In blasto-cysts, they weredetected in trophoblast, embryoblast and endoderm cells respectively.It is evident that the sperm centrosome is the functional activecentrosome in the human, while the female is inactive but maycontribute some centrosomal material to the zygote centrosome.It is very likely that the paternal centriole is the ancestorof the centrioles in fetal and adult somatic cells.     

Sequential assessment of individually cultured human embryos as an indicator of subsequent good quality blastocyst development

BACKGROUND: It is of fundamental importance for IVF clinics to determine the most viable embryos for transfer. The challenge for ART clinics is to transfer fewer embryos, thereby minimizing the risk of multiple-infant births, while still maintaining the greatest chance of pregnancy for their patients. In this study, an investigation was made to determine if developmental markers on the day of fertilization (day 1) can predict good subsequent blastocyst development. METHODS AND RESULTS: A total of 1550 individually cultured 2PN embryos from 191 patients undergoing IVF/ICSI treatment at the Yale University Center for Reproductive Medicine and Infertility from February to December 2001 was included. The results showed a significant positive relationship between early-cleaving 2-cell embryos and subsequent good quality > or =4-cell, > or =7-cell and blastocyst development (P < 0.05). PN symmetry (the relative size of the PN to each other), when checked at the time fertilization, is also a significant indictor of good quality > or =4-cell, > or =7-cell stage embryos and blastocysts. Combined, a developing embryo showing PN symmetry with early cleavage and subsequent good > or =4-cell and > or =7-cell cleavage, has a one in two chance of developing into a good-quality blastocyst. CONCLUSION: Early embryo assessment can be used as an indicator of subsequent good blastocyst development.     

The CryoLoop facilitates re-vitrification of embryos at four successive stages of development without impairing embryo growth

BACKGROUND: Vitrification has been shown to be an effective method of cryopreservation, but little is known about re-vitrification of embryos. This study investigated the effect of re-vitrification on mouse embryo preimplantation development and viability post-transfer. METHODS: Mouse embryos at the 1-cell stage were vitrified using the CryoLoop technique. Embryos were warmed and then re-vitrified successively at the 2-, 8-cell and blastocyst stages. The effects of multiple rounds of vitrification on development, differentiation and viability were assessed and compared with non-vitrified embryos. RESULTS: Development to the 8-cell stage on day 3 and blastocyst on day 5 were not affected by re-vitrification. However, better hatching rates were observed in the non-vitrified control group. Total cell number and the number of cells allocated to the inner cell mass (ICM) were not different between treatments. The percentage of ICM development was also not different between treatments. Implantation rate and fetal weights were the same between treatments. However, overall there were fewer fetuses per embryo transferred in the re-vitrified group. CONCLUSION: Re-vitrification of mouse embryos has minimal effect on preimplantation embryo development or implantation potential.     

Effects of taurine on human embryo development in vitro.

Glutamine and taurine are reported to be beneficial for mouse embryo development in vitro, and we have recently shown that glutamine improves human blastocyst formation in vitro. This randomized study compared the development of supernumerary human embryos in the presence of 1 mmol/l glutamine and/or 5 mmol/l taurine from the 2-4-cell stage to the blastocyst stage. Blastocyst development and cell numbers were similar in the presence of glutamine or taurine: 52.6% and 58.3% of the embryos reached the blastocyst stage, respectively. Pyruvate uptake was similar in the presence of glutamine or taurine throughout development, as was lactate production after the 8-cell stage. Before this stage, lactate production was 4-fold higher in the presence of taurine (P < 0.001). The proportion of embryos reaching the blastocyst stage was similar with glutamine alone or with glutamine and taurine (62.5% and 47.2% respectively), as were the blastocyst cell numbers (63.0 +/- 4.6 and 61.0 +/- 5.1 respectively). In conclusion, taurine supports development of 2-4-cell human embryos to the blastocyst stage, although it does not further augment the beneficial effects of glutamine.     

The impact of cellular fragmentation induced experimentally at different stages of mouse preimplantation development

It has been demonstrated previously that removal of acellular debris from the preimplantation mouse embryo is beneficial for subsequent development to the hatched blastocyst stage. We have studied the impact of cellular fragmentation induced in the mouse embryo during the late pronuclei and 8-cell stages on the hatching frequency and total cell number at the blastocyst stage. At the late pronuclei stage about one- quarter of the cytoplasm was removed from embryos in the experimental group, in four to six steps, thus creating four to six cytoplasts that were subsequently returned as anucleated fragments under the zona pellucida. Embryos with one-quarter of the cytoplasm removed and with intact cytoplasm after partial zona dissection (PZD) served as controls. At the 8-cell stage, embryos with their nucleoplast removed from two blastomeres served as an experimental group. Groups of embryos with part of the cytoplast removed from two blastomeres (nucleated fragments), embryos with two blastomeres removed and embryos after PZD alone served as controls. After manipulation all embryos were left in culture and analysed at about 100 h after human chorionic gonadotrophin administration. Fragments induced at the late pronuclei stage did not participate in compaction and were often spontaneously expelled from the embryo during hatching. Neither embryo hatching rate nor total cell number was affected when compared with zygotes with reduced cytoplasm. Although both nucleated and anucleated fragments induced at the 8-cell stage participated in recompaction, hatching was not compromised and there was no interference in further development as assessed by the cell number or hatching rate at the blastocyst stage, as compared with embryos with blastomeres removed. We conclude that anucleated cellular fragments formed in an otherwise healthy embryo, both before and after acquisition of the ability for compaction, are benign and that their removal provides no benefit for embryo development, at least to the hatched blastocyst stage.      

Fertilization and Empryology: The effect of epidermal growth factor on growth and differentiation of mouse preimplantation embryos in vitro

The effect of epidermal growth factor (EGF) on embryonic growth,development, attachment and spreading in vitro was studied.EGF was added to 130 embryos at the 4-cell stage; to 128 embryosat the blastocyst stage; and to 147 embryos 24 h following spreading.Development of embryos from the 4-cell to the blastocyst stage,differentiation of the inner cell mass (ICM) and trophectoderm,and the occurrence of attachment and spreading were evaluated.Embryo development was significantly inhibited in cultures supplementedwith 100 ng/ml EGF compared to the controls (P < 0.001).Development of 4-cell embryos to blastocysts occurred in 25%of the EGF group compared to 85% of controls. Spreading occurredin 20% of 4-cell embryos and 30% of blastocysts treated withEGF, compared to 80 and 90% of corresponding controls. In embryosdeveloping from the 4-cell stage, massive growth of the ICMand inhibition of the trophectoderm occurred, whereas both ICMand trophectoderm were inhibited by EGF in embryos developingfrom the blastocyst stage. Following spreading, EGF caused massivegrowth of the ICM and regression of the trophectoderm. Our preliminaryresults show that EGF may be involved in the modulation andcontrol of early embryonic growth and differentiation.     

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.     

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.     

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.     

Fatty acid metabolism in human preimplantation embryos

BACKGROUND: Little is known of fatty acid metabolism in human embryos. This information would be useful in developing metabolic tests of embryo quality and improving embryo culture media. METHODS: The fatty acid composition of human embryos and their ability to accumulate 13C labelled fatty acids was assessed in relation to the stage of development using gas-chromatography and combustion-isotope-ratio-mass spectrometry. RESULTS: Compared with embryos which did not develop beyond the 4-cell stage, those that did had significantly higher concentrations of the unsaturates, linoleic (12% versus 3%; P=0.02) and oleic (14% versus 7%; P=0.02), and a lower concentration of total saturates (62% versus 77%; P=0.04). There was uptake of both 13C linoleic and palmitic, but the developmental pattern was different for each fatty acid. The net accumulation in pmol/embryo/24h for palmitic was 1 at the 2-cell to <8-cell stage, 4 at the 8-cell-morula stage and negligible at the blastocyst stage. For linoleic, there was little net accumulation at the 2-cell to <8-cell stage, 8 (8-cell-morula stage) and 17 pmol/embryo/24 h (blastocyst stage). CONCLUSION: Preimplantation human embryos actively take up individual fatty acids at different rates at different stages of development. The high unsaturated concentration at the later stages of development may be explained by preferential uptake of linoleic acid.     

Cross-linking of Qa-2 protein, the Ped gene product, increases the cleavage rate of C57BL/6 preimplantation mouse embryos

The Qa-2 protein, a glycosylphosphatidylinositol (GPI)-linked major histocompatibility complex (MHC) Class Ib molecule found on the surface of mouse T-cells and preimplantation embryos, is the product of the preimplantation embryo development (Ped) gene. The Ped gene regulates the rate of early embryonic development and subsequent embryo survival. T-cells treated with anti-Qa-2 monoclonal antibody (mAb) and cross-linked with a secondary antibody, in the presence of a co-stimulatory signal, undergo increased proliferation. The purpose of this study was to determine whether cross-linking of Qa-2 similarly affects preimplantation embryos. We cross-linked Qa-2 protein on the surface of C57BL/6 2-cell and 8-cell embryos, in the presence of 4/5-phorbol-12-myristate-13-acetate (PMA), and assessed the percentage of embryos reaching the blastocyst stage, the percentage hatching from the zona pellucida, [(3)H-thymidine] incorporation into DNA, and the total number of cells per embryo as measures of embryonic cleavage rate. Both 2-cell and 8-cell embryos increased their cleavage rates 48 h after cross-linking of Qa-2, compared with control embryos (P < 0.05). Our results indicate that a Qa-2 protein cross-linking mechanism may be one way by which this protein regulates the rate of preimplantation mouse embryo development.     

Time from insemination to first cleavage predicts developmental competence of human preimplantation embryos in vitro

BACKGROUND: The absence of reliable markers for the identification of viable embryos for transfer at the early cleavage stage is likely to contribute to the generally low implantation rates and high incidence of multiple gestation in IVF treatment. In this study, we investigate the relationship between timing of first cleavage and the incidence of blastocyst formation in vitro. METHODS: Couples (n = 70) with at least one embryo remaining after transfer were included in the analyses. All embryos (n = 579) were examined for early cleavage at 25 h after insemination. Following embryo transfer, the remaining embryos (n = 426) were cultured until day 7 of development, and assessed for blastocyst formation. RESULTS: Eighty-five embryos (14.7%) cleaved to the 2-cell stage within 25 h of insemination; 26 of these were selected for transfer on day 2. Of the 59 embryos remaining in culture, 19 (32.2%) developed to the blastocyst stage; this was a significantly higher number than was observed in embryos (61/367; 16.6%) that failed to cleave within 25 h of insemination (P < 0.01). Within these two groups of embryos the proportion of hatched blastocysts was 11/59 (18.6%) and 26/367 (7.1%) respectively (P < 0.005). CONCLUSIONS: These findings indicate that early cleavage is indicative of increased developmental potential in human embryos and may be useful as an additional criterion in the selection of embryos for transfer.     

植入前诊断中胚胎活检的动物实验研究

目的以小鼠胚胎为材料进行植入前胚胎活检的研究.方法通过输卵管冲洗,获得4～8细胞期胚胎,用"一"字机械法、化学法和激光法分别对4、8细胞的胚胎进行活检,观察其囊胚形成及孵出情况并进行比较.结果 "一"字机械法、化学法、激光法胚胎活检后胚胎的囊胚形成率、囊胚孵出率与未活检对照组比较无显著性差异(P>0.05). "一"字机械法与化学法、激光法的囊胚形成率有显著性差异(P<0.05)、囊胚孵出率无显著性差异(P>0.05).化学法与激光法的囊胚形成率、囊胚孵出率均无显著性差异(P>0.05).logistic回归分析表明:细胞数越多越有利于胚胎进一步发育.结论用"一"字机械法进行单个卵裂球活检,不影响胚胎的进一步发育,是一种较好的胚胎活检方法.     

The effect of intracytoplasmic sperm injection and semen parameters on blastocyst development in vitro

The present study compares the development and quality of blastocysts derived from conventional oocyte insemination with those derived from intracytoplasmic sperm injection (ICSI). Oocytes were collected from patients undergoing ovarian stimulation with human menopausal gonadotrophins for IVF. Patients with normal semen were assigned to conventional oocyte insemination while those with progressive motility <20% and/or normal sperm morphology < or =4% were assigned to ICSI. Resulting embryos were cultured for up to 6 days. The mean number and percentage of embryos reaching the blastocyst stage and the mean number and percentage of blastocysts of high quality on days 5-6 were assessed for both treatment groups and compared. The influence of paternal factors (sperm concentration, motility, progressive motility, morphology) on blastocyst development and quality were assessed by regression analyses. Significantly more ICSI-derived embryos arrested at the 5- to 8-cell stage (P = 0.024) concomitant with the activation of the paternal genome than those derived from conventional oocyte insemination. Significantly fewer ICSI-derived embryos reached the blastocyst stage on days 5-6 (P<0.001) and significantly fewer ICSI-derived embryos were of high quality (P = 0.002) compared with conventional oocyte insemination. When treatment groups were combined and evaluated by regression analysis, progressive motility and sperm morphology were significantly correlated with diminished blastocyst development and quality (P < 0.05). From these data, we conclude that paternal factors and/or performing ICSI in cases of severe male factor infertility may have a detrimental effect on blastocyst development and their quality.     

Fertilization and early embryology: Effects of pentoxifylline on in-vitro development of preimplantation mouse embryos

In-vitro culture of 1-cell mouse embryos was used to assessthe influence of pentoxifylline on early embryonic development.If cultured in concentrations of 5, 10 or 50 µM, earlyembryonic development was unaffected and no differences in cellnumbers were noted in embryos reaching the blastocyst stage.However, at 3.6 and 7.2 mM, pentoxifylline inhibited cleavagefrom the 2-cell stage onwards. If 1-cell mouse embryos wereexposed for only 30 min to these concentrations, blastocystformation was found to be morphologically normal. However, cellnumbers of such blastocysts were significantly decreased afterexposure to pentoxifylline. These results may indicate thatexposure of gametes or zygotes to pentoxifylline should be avoidedas much as possible when this drug is used in human assistedreproduction. If administered at regular therapeutic doses,it is probable that no adverse effect on early embryonic developmentin vivo will occur. Further research is needed to confirm andelucidate the above findings.     

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.     

Improved development of human embryos in vitro by a human oviductal cell co-culture system.

This study was undertaken to determine the effect of co-culture with human oviductal cells on human embryos. Spare embryos from gamete intra-Fallopian transfer (GIFT), pronuclear stage transfer (PROST) and in-vitro fertilization/embryo transfer (IVF/ET) programmes were either cultured in serum-supplemented Earle's balanced salt solution alone, or co-cultured in the same solution with oviductal cells from the pronuclear stage (day 1 post-insemination) or two- to four-cell stage (day 2 post-insemination). The co-cultured embryos appeared to have a higher developmental potential (higher rate of blastocyst formation and lower fragmentation rate), although there was no statistical difference in their rate of development, degree of fragmentation and stages attained, when compared with conventionally cultured embryos. The percentage of hatching blastocysts was significantly higher (P less than 0.05, Fisher's exact test) for embryos co-cultured from day 1 post-insemination (38%) than for embryos which had not been co-cultured (7%). The blastocyst hatching rate for embryos co-cultured from day 2 post-insemination was 15%. It was therefore concluded that co-culture of human embryos with oviductal cells could improve the development of the embryos in vitro. The degree of improvement was more pronounced when the co-culture started at an earlier stage.     

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.