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Mouse in vitro fertilization, embryo development and viability, and human sperm motility in substances used for human sperm preparation for assisted reproduction
Reproductive animal research FERTILIZED
STERILIZE
Februaxy
Vol.67,No.2,
Mouse in vitro fertilization, embryo development and viability, human sperm motility in substances used for human sperm preparation for assisted reproduction* Lynette Samuel
199:
and
Scott, M.Sc.t Smith, M.D.
The Assisted Gy31ecology,
Reproduction Sin.ai Hospital,
Technologies Baltimore,
Program, Ma~~~~d
Women’s
Health
Center
at Sinai
Hospital,
L3epnrtmen.t
of Obstetrics
and
To investigate the endotoxin content and effects on mouse IVF and embryo develand human sperm motility of human sperm separation substances. De&u: One-cell and zona-free two-cell mouse embryo bioassays and Limulus Amoebocyte Lysate endotoxin tests and mouse oocyte parthenogenetic activation, IVF, preimplantation and postimplantation embryo development, and hnman sperm motility were performed in control medium or medium containing Percoll, Nicodenz, or the washings from Sperm Prep scphadcx columns. Setting: Research Laboratories, Sinai Hospital of Baltimore, Baltimore, Maryland. Main Outcome Measure(s): Endotoxin levels, embryo development, and sperm motility. Result(s): Mouse embryo bioassays indicated negative endotoxin levels in Percoll and Nicodenz but Limulus Amoebocyte Lysate assays had positive gel formation. Mouse IVF and preimplantation development was equivalent in control and Percoll- and Nicodenz-containing medium; none had parthenogenetic properties hut postimplantation development was reduced for embryos grown in the presence of Percoll or Nicodenz. Human sperm remained motile in the presence of Percoll or Nicodenz. Sperm Prep column washes were toxic to mouse gametes and embryos and human sperm and had gel formation with the Limulus Amoebocyte Lysate assay. Conclusion(s): Percoll and Nicodenz, but not Sperm Prep sephadex column washes, were compatible with mouse preimplalltation and postimplantation embryo development and human sperm motility. Fertil Sterile 1997;67:372-81 Objective:
opment
Key
Words:
Sperm
separating
substances,
Percoll,
The recovery of morphologically normal, intact, motile sperm from semen is required for use in most assisted reproductive techniques (1,2X The exception to this is direct intracytoplasmic sperm injection in which motile, nonmotile, immature, or even round sperm have been used for assisted fertilization (3-5).
Received April 25, 1996: revised and accepted July 29, 1996. * Presented in part at the 51st Annual Meeting of the American Society for Reproductive Medicine, Seattle, Washington, October 7 to 12, 1995. f Reprint requests: Lynette Scott, MSc.. Assisted Reproductive Technologies Program, Women’s Health Center at Sinai Hospital, 2411 West Belvedere Avenue, Weinberg Building, Suite 206, Baltimore, Maryland IFAX: ~10-578-8576~.
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mad embryo
bioaesays
endotoxins,
gamete
and embryo viability
There are a variety of sperm isolation protocols available to separate motile sperm froIm the remainder of the ejaculate. A simple method is direct swimup from the whole ejaculate. This method has proved to be inadequate in instances in which there is low density or motility because the efficiency of recovery is very low. Another widely used method has been double wash and swim-up in which the whole ejaculate initially is pelleted to remove the seminal fluid followed by swim-up from this pellet. However, centrifugation and pelleting of unselected sperm samples can have an adverse affect on sperm. This primarily is due to membrane damage caused by superoxide radicals produced by neutrophils and other cells that are pelleted during centrifugation
Fertility
and
Sterilitya
(6-8). Repetitive centrifugation also mechanically can damage sperm, leading to decreased survival, motility, and function (9). An alternate approach is to separate the motile intact sperm from the rest of the ejaculate before pelleting. There are a number of ways to accomplish this, generally involving centrifugation on a density gradient made from polyvinylpyrrolidone-coated silica beads (Percoll, Nicodenz, or Ficoll) or migration through a sephadex matrix (Sperm Prep column) or glass wool. However, migration of the sperm through the separating substance can leave the sperm either coated with particles from the separation column (10) or mixed with some of the separating substance. Additional washing, and therefore centrifugation, of the sperm sample to eliminate these contaminants can induce damage (9). Furthermore, there have been reports of endotoxin contamination in certain of these products when using a Limulus Amoebocyte Lysate Test (Svalander PC, Lundin K, Holmes PV, abstract). Because endotoxins have been shown to be associated with increased fragmentation of human embryos (11) and nonviability of mouse embryos (12), it is thought that the sperm require washing, with centrifugation, to remove any traces of these substance before use in assisted fertilization techniques. In cases in which there is extremely low sperm density and/or motility, being able to use the separated sperm sample without additional washing and centrifugation would be beneficial for microdrop insemination (13) or assisted fertilization procedures (3, 4). Our laboratory uses the one-cell and zona-free two-cell mouse embryo bioassay for quality control (12). Using these tests and in other culture experiments there has been no evidence of endotoxin contamination in Percoll. Because previous reports of endotoxin contamination in Percoll used the Limulus Amoebocyte Lysate test, we screened Percoll for endotoxin content with both the mouse embryo bioassay and commercially available Limulus Amoebocyte Lysate tests. We further screened two other sperm separation substances, Nicodenz and Sperm Prep sephadex columns. The compatibility of the three separation substances with mouse IVF in vitro embryo development and embryo viability and human sperm motility in vitro also was studied as further bioassays. MATERIALS
AND
Sinai Hospital (Protocol Number Scott/Mou/l l-93). Mice were purchased from Charles River (Wilmington, VA). A colony of proven fertile CD-11males was maintained either for mating purposes or for sperm collection for IVF. Only fertile males with r6 months of successful mating were used for sperm collection for IVF. Virgin CD-l females at 4 to 5 weeks of age were superovulated by administration of 5 to 10 IU pregnant mare serum gonadotropin (G-4877; Sigma Chemical Company, St. Louis, MO) and chorionic gonadotrophin (CG-5) was given 44 to 48 hours apart. Females designated for mating .were placed individually with males overnight and checked the following morning for the presence of a vaginal plug as evidence of successful mating. A colony of vasectomized Fl (C57BV6NCr x C3W HeNCr) males were maintained for the induction of pseudopregnancy. Virgin CD-l females, 8 to 10 weeks old, were mated with the vasectomized males and checked for vaginal plugs. The day of plug was designated day 1 of pseudopregnancy. Media
METHODS
Mice
All protocols involving mice were approved by the Institutional Animal Care and Use Committee at
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67, No. 2, February
1997
Scott
All media were made from salts and tissue culture water (W-500) purchased from Sigma. The media used for all experiments were based on a modified Earle’s Balanced Salt Solution (EMO) containing no glucose or inorganic phosphate, with added ethylenediaminetetraacetic acid (0.11 mM) and glutamine (1.0 mM). The EM0 has been shown to support >80% fully expanded blastocyst development from the one-cell stage of a number of random and inbred strains of mice (12, 14). Variations of this medium containing either 0.5 mM glucose (EMG/0.5), 2.78 mM glucose (EMG), or 2.78 mM glucose and 1.32 mM inorganic phosphate (NaPOJ (EMGP) were also prepared (12,14). Basic medium (EMO) with 0.5 mM glucose (EMG/0.5) has been shown to give optimal development of CD-l embryos from the one-cell to blastocyst stage (Scott and Whittingham, unpublished data). All media were supplemented with 3 mg/mL bovine serum albumin (BSA, A-!3647; Sigma) unless otherwise stated. Media were prepared from concentrated stock solutions as required (12, 14); the osmolarity was adjusted to 280 to 290 mOsm, gassed with 5% COZ, 5% 02, 90% Nz for 5 minutes, sterilized with a 0.22pm filter, and placed in the incubator :for overnight equilibration. Solutions of 5%, lo%, 20%, 40%, and 80% Percoll (P-1644; Sigma) or Nicodenz (227510; Nicomet Pharma AS, Oslo, Norway) in EMG10.5 were formulated by substituting a percentage of the water content with undiluted Percoll or Nicodenz.
and Smith
Sperm
preparation
and
embryo
bioassays
373
Two lot numbers of Percoll and one of Nicodenz were used. Sperm Prep columns (ZLB Inc., Lexington, KY) were washed with 4 mL of EMG/O.5, which was collected in a sterile test tube. This initial 4 mL of media (SPCl) was the volume specified by the manufacturer as a prewash but was found to be highly toxic to mouse embryos. Thus, a series of washings were carried out in which four additional 2-mL washes were collected (SPCB, SPC3, SPC4, and SPC5), for a total of 12 mL of washing medium. For each of the three repeated experiments, three columns were used and the washings were pooled. There were two lot numbers of columns used. Embryo
Collection
and Culture
Superovulated, plugged CD-l females were killed at 20 hours after hCG. Pronuclear embryos were released from the oviducts directly into warmed EM0 containing 2 mg/mL hyaluronidase in order to remove the cumulus masses. The embryos were collected and washed through three 2-mL rinses of EM0 and two lOO-PL rinses of the medium in which they were to be cultured. Embryos were placed in groups of 10 per lo-p,L drop of medium under an oil overlay in a humidified, 5% COz in air incubator for culture. No HEPES buffer was used during oocyte or embryo manipulations to limit the number of environments the embryos were subjected to and to avoid intracellular pH changes that low bicarbonate can cause, which is known to reduce development in mouse and hamster embryos (15, 16). The number of embryos continuing to cleave and appear normal was recorded. An embryo was scored as surviving if it had at least two cells at 48 hours, four cells at 72 hours, eight cells at 96 hours, and was a blastocyst at 120 hours after hCG. All other embryos were scored as nonsurvivors as were those that were fragmenting. Endotoxin
Assays
Two commercially available endotoxin kits were used (BioWhittiker, Inc, Walkersville, MD; and TGF-1, Sigma). Both were based on a timed gel formation in the Limulus Amoebocyte Lysate test and were performed as per manufacturer’s instructions. Briefly, a concentration curve of both a known endotoxin and the sample were made and tested in the system. The test is based on the observation that as the levels of endotoxin increase the time for gel formation decreases and gel hardness increases when measured against the known incubation times for increasing concentrations of the standard. The last dilution of standard at which gel formation is
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observed after 1 hour of incubation is the positive endpoint. One-cell and zona-free two-cell mou.se embryo bioassays were performed (12, 17). In these tests the media were supplemented with 3 mg/mL polyvinyl alcohol (PVA, P-8136; Sigma) and not BSA. Embryos were collected as described in embryo collection and culture, except that two-cell embryos were flushed from the oviduct at 44 hours after hCG. These were washed through two 2 mL rinses of EMG/0.5 and then placed in acid-EMG/0.5 (EMG/0.5 made acidic, pH 3, by the addition of hydrochloric acid) for up to 5 seconds. They then were washed rapidly through four 2-mL rinses of EMG/0.5 before being placed in EMGi0.5 supplemented with 3 mg/mL PVA for culture. For all the endotoxin tests, a control sample of medium containing 1 ng/mL of Pseudomonas aeruginosa endotoxin (L7081; Sigma) and nlo BSA or PVA also was tested. The endotoxin content of all media used in the experiments (EMG/0.5 with and without BSA and PVA; EMG10.5 with added endotoxin; each of the sperm separation substances) was assessed using each of the endotoxin tests described above. Experiments were repeated three times for each system. Parthenogenetic
Activation
If oocytes are to be placed into any substance other than culture medium, the artificial activating properties of that substance should be known. Unfertilized mouse oocytes surrounded by the cumulus mass were released from the oviducts at 1.5 to 16 hours after hCG directly into the medium being tested. Test media were EMG/0.5 with 20%, 40%‘, or 80% Percoll or Nicodenz and SPCl, SPCS, SPC3, SPC4, and SPC5. They were incubated in the,se media for 6 hours and then released from their cumulus masses with hyaluronidase. Activation was carried out with the cumulus mass intact to simulate the conditions of human oocyte-cumulus complexes d.uring IVF. After the activation period, oocytes were scored for the extent of parthenogenetic activation (18, 19). Oocytes activated with 10% ethanol for 5 minutes or incubated in medium alone were used as activation controls (12, 19). Once scored, the oocytes were placed in culture and scored again the following day for any signs of activation and cleavage. Experiments were repeated three times with at least two cumulus masses per treatment in each repeat. In vitro
Fertilization
Superovulated females for oocyte collection were killed 12 to 14 hours after hCG and males were killed 24 hours before the IVF to allow the sperm to capac-
Fertility
and
Sterility3
itate. After killing the male mice, the epididymis was excised and freed of as much of the connective tissue and blood vessels as possible, using sterile instruments. The contents of the epididymis were squeezed out, using sterile watchmakers forceps, into 2 mL EMGP supplemented with 10 mg/mL BSA and placed in the incubator at 37”C, with 5% CO2 in air. After 4 hours, the medium was aspirated gently out of the dish and the sperm concentration ascertained using a Makler counting chamber. The sperm were diluted with EMGP to approximately 2 x lo61 mL and then further diluted to 0.75 to 1.0 x lo6 motile sperm/ml with control medium or medium containing 40% Percoll, 40% Nicodenz, or the fourth washing from the Sperm Prep Column (SPC4). This resulted in approximately 20% Percoll and Nicodenz and half the amount of SP4 in the insemination medium. Drops of 100 I.~L of this sperm suspension were placed under oil as insemination drops. All oocyte and embryo manipulations were carried out under equilibrated mineral oil (M-3516; Sigma) on a heated microscope stage that was maintained at 36 to 37°C (12, 14). Oocyte-cumulus complexes intended for IVF were released from the oviduct into EMO, picked up with a flame-polished Pasteur pipette, and placed directly into the 100 /IL insemination drops. After 6 hours, 100 PL of EMG/0.5 with 2 mg/mL hyaluronidase (H-3506; Sigma) was added to the insemination drops to release the oocytes from their cumulus masses. The oocytes were washed with three 2-mL rinses of EMG/0.5 and then placed in lo-PL drops of this medium under oil. The oocytes were scored on an inverted microscope with Hoffman differential contrast optics at magnifications of X 3 10 and x500 for signs of fertilization, which were the presence of two polar bodies and two pronuclei. The number of oocytes fertilized in experimental media was compared with the number fertilized in control medium (EMGP). Fertilized oocytes were moved to EMG/0.5 for further development and grown for 100 hours (120 hours after hCG) as described above in embryo culture. The cell numbers in expanded blastocysts were obtained as described previously (14). Experiments were repeated three times with at least three cumulus masses per treatment group in each repeat. In Vitro
Embryo
tially, it was found that all embryos arrested at the morula stage when grown in 40% or 80% Percoll or Nicodenz. These embryos floated at the surface of the medium drops, at the oil-medium interface. If they were moved to 10% Percoll or Nicodenz after the initial 48 hours of culture, they continued growing. Thus, for the data presented for the 40% or 80% Percoll or Nicodenz, the embryos were grown for the first 48 hours in these concentrations and then moved to 10% Percoll or Nicodenz, respectively, for continued culture. All results were compared with control embryos cultured in EM0 with 0.5 mM glucose (EMG/0.5). Cell numbers in expanded blastocysts were obtained at 120 hours after hCG as described previously (15). Experiments were repeated three times with 20 embryos per group for each repeat. Embryo
The effect of exposing embryos to Percoll, Nicodenz, or SPC washes on their ability to implant and develop in pseudopregnant females was tested. For this, pronuclear embryos were cultured in 20% Per~011,20% Nicodenz, SPC4, or control medium for 72 or 88 hours. They were transferred to pseudopregnant females on the afternoon of day 3 of pseudopregnancy as morulae (96 hours after hCG) or blastocysts or on the morning of day 4 as expanded blastocysts (112 hours after hCG) (14). Elxperimental embryos were transferred to one uterine horn of pseudopregnant females and control embryos, grown in EMG/0.5, and transferred to the other. Five or six embryos at approximately the same developmental stage were transferred to each horn, thus making the choice of embryos for transfer nonrandom. This was to remove any implantation advantage any one group would have over another. However, the number of implantation sites and live pups on day 18 of pseudopregnancy were compared within a mouse (experimental versus control; paired t-test) and then overall between media (x2 analysis) for differences. Human
67, No. 2, February
Development
1997
Sperm
Preparation
and Motility
Sperm samples were collected by masturbation and allowed to liquefy for 30 minutes at 37°C. A basic semen analysis was performed (volume, density, motility). The sample then was separated on a two-step (40%, 80%) mini-Percoll (0.3 ImL) gradient (13). After centrifugation at 250 x g for 20 minutes, the upper layer of 40% Percoll and semen were removed. The sperm pellet then was aspirated off the bottom of the tube using a sterile Pasteur pipette. This pellet was placed in a centrifuge tube with care being taken not to touch the inside of the tube with
Pronuclear embryos were collected at 20 hours after hCG as described above and placed in different media for development over 96 hours (120 hours after hCG). The treatment groups were EMGj0.5, 5%, lo%, 20%, 40%, and 80% Percoll and Nicodenz and each of the Sperm Prep column washings. Ini-
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375
the outside of the pipette. The sperm pellet was suspended in 2 mL basic medium with 2.78 mM glucose (EMG) and aliquoted into eight 0.4-mL volumes. The aliquots were resuspended in 2 mL of EMG containing 20% or 40% Percoll, 20% or 40% Nicodenz, and 100% of each of SPCl, SPC3, SPC5, or control medium. They were centrifuged for 5 minutes at 250 x g and the final pellet was resuspended in 0.5 mL of the medium to which they were designated. The percent of motile sperm was then assessed after 8 and 24 hours of incubation at 37°C and compared with the motility in the control medium. The data were analyzed between media within a sample (t-test and analysis of variance [ANOVAI) and overall between the media. RESULTS Endotoxin
Contamination
The CD-l embryos showed no signs of fragmentation in either a one-cell or zona-free two-cell mouse embryo bioassay when cultured in control medium or medium with 20% Percoll or 20% Nicodenz (Fig. 1). In all these treatments there was >70% expanded blastocyst formation by 120 hours after hCG, which compares favorably with results previously reported for CD-l embryos in EMG (12, 14). In contrast, mouse embryo development from the one-cell and two-cell stage in SPC washes was impaired severely. In both bioassay systems, all embryos became atretic within an hour of being placed in SPCl, SPCB, or SPC3. There was limited development and
survival in the SPC4 and SPC5. Thfe embryos became dark and atretic but did not display any fragmentation and membrane perturbations that are seen with endotoxin contamination and in endotoxin-spiked medium. Control medium containing 1 ng/mL of endotoxin resulted in 0% blastocyst formation, cell membrane perturbations, extreme fragmentation, and developmental arrest, Using the Limulus Amoebocyte Lysate kits, control medium (EMG/0.5) supplemented with BSA or PVA was negative for endotoxin. Control medium spiked with 1 ng/mL endotoxin resulted in gel formation in both test kits at a level indicating approximately 1 ng/mL endotoxin (data not shown). The Limulus Amoebocyte Lysate kits worked consistently well with concentration curves of endotoxin and on other products. However, both Percoll and Nicodenz had gel formation in both Limulus Amoebocyte Lysate kits. The time of gel formation and the extent of gelling was extremely variable, dependent on the concentration of either substance and the buffer used as diluent (data not shown). Sperm Prep column washings had gel form.ation on both test kits. The extent of gelling decreased with washing number. Parthenogenetic
Normally aging untreated oocytes spontaneously activate at very low levels in vitro (5%; 71147 oocytes from seven cumulus masses) (data not shown). Control ethanol activations resulted in 58% activation (88/152 oocytes from eight cumulus masses) with 49% (43/88) of activated oocytes cleaving to at least the eight-cell stage. The level of activation in 20% Percoll(6%; 8/134, six cumulus masses’),40% Percoll (3%; 4/123, five cumulus masses), 80% Percoll (4%; 5/119, five cumulus masses), and 20% Nicodenz (5%; 5/107, four cumulus masses), 40% Nicodenz (1%; l/112, five cumulus masses), and 80% Nicodenz (7%; g/126, six cumulus masses) was not significantly greater than that obtained with aging untreated oocytes. Sperm Prep column washings SPCl, SPCB, and SPC3 did not activate oocytes; they caused the oocytes to become dark and atretic in appearance whereas SPC4 and SPC5 resulted in no activation and minimal atresia in the oocytes (data not shown). In Vitro
Figure 1 Data represent the mean 2 SEM of three replicate mouse embryo bioassays for endotoxin using the one-cell and zona-free two-cell tests. All media systems were tested in each repeat experiment.
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Activation
Fertilization
There were no differences in the percentages of oocytes fertilized in control medium or medium containing 20% Percoll or 20% Nicodenz (Table 1). The exposure of sperm and oocytes to a 1 :l dilution of SPC4 resulted in a decrease in the number of oocytes
Fertility
and
Sterilitys
Table 1 IVF of CD-l Oocytes With Sperm Prepared With of the Resulting Embryos for 100 Hours in Embryo Culture
Control Medium
(EMGP) and Experimental (EMGIO.5)” Medium
Control No. No. No. No. Cell
of cumulus masses of oocytes of fertilized oocytes$ of blastocytes$ no. in blastocystsl
medium?
Table 2 The In Embryos Grown Pronuclear Stage (120 Hours After
1997
9 iii (49 9.3)s 29 (60: 4.7)1/ 42 (8.4)*”
Vitro Development and Cell in Control and Experimental (20 Hours After hCG) to the hCG) From Three Repeated
Number of CD-l Media From the Blastocyst Stage Experiments
Blastocyst developmentt
Culture system”
Development
67, No. 2, February
10 108 68 (63, 6.2) 51 (75, 4.1) 60 (3.9)
There was no survival of CD-l embryos in the first three SPC washes. The SPC4 and SPC5 resulted in better survival but this was significantly less than controls (P < 0.001). The embryos in t.hese experiments became atretic, fragmented, and did not
There were no differences in blastocyst development of CD-1 mouse embryos grown in control medium or with 5%, lo%, or 20% Percoll or Nicodenz (Table 2). Higher concentrations of Percoll and Nicodenz resulted in a slight decrease in blastocyst formation (P < 0.05). Development of embryos in 80% Nicodenz for the first 48 hours was reduced significantly compared with development in lower concentrations of Nicodenz or Percoll or in control medium (P = 0.001, paired t-test and ANOVA). The cell numbers in control embryos and in those cultured in 5%, lO%, and 20% Percoll or 5% or 10% Nicodenz were not significantly different. There was a reduction in the cell numbers of embryos grown in 20% Nicodenz (P < 0.05). There was a reduction in cell number of embryos initially grown in 40% Perco11(P < 0.05) or 40% Nicodenz (P < 0.01) and of those initially grown in 80% Percoll (P < 0.001) or 80% Nicodenz (P < 0.001) compared with control medium.
Vol.
0.5 x SPC4’F
0 P < 0.0005 versus control. 11P < 0.005 versus control. n Values in parentheses are SEM. **P < 0.001 versus control,
fertilized (P < 0.005, t-test and ANOVA). The mouse sperm in these insemination drops had decreased motility and survival compared with that seen in control medium, Percoll-, and Nicodenz-containing medium. The development to blastocysts was equivalent for embryos fertilized in insemination medium made from control, Percoll, and Nicodenz but was reduced for embryos resulting from 0.5 x SPC4 insemination drops (P < 0.005). The cell numbers in expanded blastocysts after 100 hours of culture followed a similar pattern. There were no differences in the cell numbers of embryos resulting from insemination in control, Percoll, or Nicodenz insemination media. There was a significant reduction in cell number in embryos resulting from insemination in a 1:l dilution of SPC4 insemination medium (P < 0.001). Embryo
20% Nicodenzt
12 114 74 (65, 4.7) 52 (70, 1.91 58 (2.7)
* Data are the sum of three repeat experiments, t All IVF was in EMGP and subsequent embryo culture was in EMGI0.5. f Values in parentheses are percentages and SEM, respectively.
and the Devel,opment
system
20% Percollt
9 97 65 (67, 3.4) 47 (72, 2.8) 61 (1.8)
Medium
Scott
EMGI0.5 Percoll 5% 107c 20% 40% 80% Nicodenz 5% 10% 20% 40% 80% Sperm prep SPC-1 SPC-2 SPC-3 SPC-4 SPC-5
Total embryos cultured
180
Blastocyst cell no.+
Mean ? SEM
Tota I embryos counted
Mean ? SEM
83 5
2.7
30
66 -t 2.3
60 60 60 60 60
72 75 77 63 62
+ t 2 + i
2.5 2.3 2.9 2.98 2.91,
30 30 30 30 30
65 67 64 56 43
k 2 + k t
1.9 1.5 1.5 1.69 2.1(1
60 60 60 60 60
77 69 69 63 35
+ 3.4 2 5.1 2 4.3 ? 10.95 5 5.61
30 30 30 30 30
66 58 56 47 34
I i -+ + 5
2.2 2.1 1.85 1.911 2.31
0 0
-
37 2 2.3$ 44 -t 2.517
60 60 60 60 60
5 x 40 2 52 2
1.g**: 7.11 3.01
14 20
* Culture system in which the embryos were grown. t Percent blastocyst and expanded blastocyst development at 120 hours after hCG. $ Cell number in blastocysts and expanded blastocysts at 120 hours after hCG. 5 P < 0.05 versus controls. I/P < 0.01 versus controls. ll P < 0.001 versus controls. **p < 0.0001.
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Table 3 The Implantation and Fetal Development of CD-l Embryos at 18 Days of Gestation That Were Cultured (Control) or in EMG/0.5 Containing 20% Percoll, 20% Nicodenz, or SPC4 (Experimental) for 76 or 92 Hours (96 and 112 Hours After hCG) 20% Control No. of pregnant recipients No. of embryos transferred No. of embryos implanted No. of live fetuses Implantation* (%) Live$ (%‘o)
Percoll
* Percent of transferred embryos tP < 0.05 versus EMGI0.5. $ Percent of transferred embryos
20% Nicodenz
Experimental
6 33 26 18 79 55
Control
6 33 22 16
Control
6 33 18 3 551 3%
3 P < 0.01 versus 11P < 0.001 versus
4 17 11 10 65 59
4 15 3 2
2Oll 1311
EMG/0.5. EMG/O.S
hours resulted in a decrease in motility compared with control medium (P < 0.05). After 24 hours, the motility was reduced further for both Percoll (P < 0.01) and Nicodenz (P < 0.001).
Viability
There was no difference in the implantation rates of morulae or blastocysts grown in 20% Percoll or control medium (Table 3). There was a slight but significant decrease in the number of live fetuses seen with embryos grown in 20% Percoll. Both the implantation (P < 0.05) and the number of live fetuses (P < 0.01) were reduced when embryos were grown to the morula and blastocyst stage in Nicodenz compared with those grown in control medium. There was very poor implantation and fetal development of morulae and blastocysts grown in SPC4 compared with control medium and Percoll and Nicodenz cultures.
8 Hours
I *
*** ai**
EMG
20%
40%
PWCOII
Human Sperm Substances
Motility
in Sperm
Separation
The motility of the sperm samples at 8 and 24 hours of incubation in control (EMG) and experimental media are shown in Figure 2. In control medium, there was a decrease in the percent of motile sperm with time that was sample dependent. Some individual samples had a significant reduction in motility after both 8 and 24 hours of incubation. A similar pattern was seen for each individual sample in both Percoll and Nicodenz, thus the data is presented as the mean + SEM survival. Incubation in 20% Percoll or 20% Nicodenz for 8 hours did not significantly alter the motility of the sperm samples when compared with the same samples in test medium. However, after 24 hours incubation, there was a reduction in motility in both 20% Percoll (P < 0.01) and 20% Nicodenz (P < 0.001) compared with control medium. Incubation in 40% Percoll or 40% Nicodenz for 8
378
Experimental
in a live fetus.
cleave. The cell numbers in embryos grown in SPC4 and SPC5 were significantly less than those grown in control medium (P < 0.001). Embryo
SPC-4
Experimental
6 34 23 17 68 50
implanting. resulting
in EMG/0.5
Scott
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Sperm
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embryo
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loo 75
hi !O%
40%
SFCI
SC3
WCS
NlCCKh2
24 Hours
-Al
0 EMG
20%
e
20%
40%
WC1
SK3
spcs
Figure 2 Data represent the mean -t SEM motility at 8 and 24 hours of 10 samples in control (EMG) and experimental media. Each sample acted as its own control. Patterns of motility were the same for each sample in the different media, although the samples were different from each other. The data were analyzed within a sample and then overall for media differences. Control medium was EMG, which was the diluent for Percoll, Nicodenz, and SPC washes. *P < 0.05; **P < 0.01; ***P ,< 0.001.
Fertility
and
Sterility@
Sperm motility in SPCl after 8 hours incubation was decreased significantly compared with control medium, Percoll, and Nicodenz (P < 0.0001). After 24 hours incubation, there were no motile sperm in SPCl. Sperm motility in SPC3 was slightly better but still significantly less than that obtained with control medium, Percoll, and Nicodenz at both 8 and 24 hours of incubation (P < 0.001). The final washing, SPC5, resulted in sperm motility that was reduced at 8 hours of incubation (P < 0.05) and 24 hours of incubation (P < 0.0001). However, these data were no different to those obtained with 40% Percoll and Nicodenz at 24 hours and 20% and 40% Nicodenz at 24 hours of incubation. DISCUSSION
The results presented here indicate that polyvinylpyrrolidone-coated silica bead density gradient sperm separation substances such as Percoll and Nicodenz are compatible with mouse oocytes IVF and embryo development. However, there was reduced viability of embryos grown for extended periods in these substances at a concentration of 20%. Human sperm remained motile in 20% Percoll and Nicodenz for 24 hours. In contrast, the wash medium from a Sephadex-based sperm separation column was highly toxic to mouse oocytes, embryos, and human sperm. The toxic substances washed off the columns appeared to decrease with multiple washes but a significant cytotoxic effect was still noted after 12 mL of wash, which was equivalent to six column volumes of wash. The presence of endotoxins in embryo culture media and on other embryo contact materials is harmful to embryos. Mouse (12, 17) and human embryos (11) both exhibit increased fragmentation and reduced viability in the presence of endotoxins. In this study, endotoxin contamination was not found in either Percoll or Nicodenz using the mouse embryo bioassay. It has been reported that Percoll has high concentrations of endotoxins (Svalander PC, Lundin K, Holmes PV, abstract) when tested with a Limulus Amoebocyte assay. In these experiments, gel formation, which is consistent with endotoxin contamination, was found with in both of Percoll and Nicodenz when using a Limulus Lysate Assay. Because there was no evidence of endotoxins with the mouse embryo bioassay, it was speculated that this gel formation was an artifact. The use of the Limulus Amoebocyte Lysate assay to test for endotoxin contamination in raw products has been reported to be problematic due to poor specificity. It initially was found that only a small percentage of substances initially screened could be
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tested without modifications. This was due to either inhibitory or enhancement mechanisms, which resulted in false-positive or -negative tests (20-22). There are many conditions that can affect the Limulus Amoebocyte Lysate reaction, which is a cascade of serine protease reactions triggered by endotoxin and finally resulting in the formation of a gel. The reaction is sensitive to pH; the presence 1orabsence of divalent cations; the presence or absenoe of cellulose materials that contain Limulus Amoebocyte Lysatereactive material, typically B-glycans; oxidants or antioxidants that can affect the protease reactions; and serum proteolytic enzymes. The reaction mix has slight buffering capacity between pH 6 and pH 8. Enhancement of the reaction can occur if the concentrations of either or both Mg2+ and Ca2+ are not optimized. When tested in the rabbit pyrogen test, most Limulus Amoebocyte Lysate-reactive material is negative and does not cause any other in vitro effects. Percoll and Nicodenz are colloidal suspensions of silica coated with polyvinylpyrrolidone. They have a room temperature pH of 9.0. Both will spontaneously gel as the pH is lowered. It was found that simply lowering the pH of Percoll to 6.6 and Nicodenz to 6.0 resulted in spontaneous gelling (data not shown). Certain buffer systems also caused gelling of Percoll and Nicodenz. The gelling effect was augmented by an increase in temperature and with increased concentrations of Percoll and Nicodenz. Further, when 100% Percoll was mixed with the Limulus Amoebocyte Lysate reaction mix (1:5 ratio), the resulting pH was 6.8. If a bicarbonate (Earle’s blalanced salt solution) or a phosphate buffer initially was used to dilute the Percoll to 50%, the pH was decreased to 6.2. In both instances, there was gel formation within seconds of adding the Limulus Amoebocyte Lysate reaction ingredients to the tube. These data suggest that the positive Limulus Amoebocyte Lysate endotoxin test seen with Percoll and Nicodenz could in fact be false positives related to the combined effects of the properties of both the Limulus Amoebocyte Lysate test and the nature of Percoll and Nicodenz. The absence of i:nflammatory and other cellular responses when Percoll was injected directly into the uterine horns or ovaries of rabbits supports this contention (23). The toxicity of the wash medium from the Sperm Prep columns on mouse embryos and the positive reaction on the Limulus Amoebocyte Lysate tests made it hard to draw conclusions as to the endotoxin content of these columns. The mouse embryo toxicity could be due to any preservative, buffer, or fungicide that may be in the Sephadex to preserve the columns before use, These substances also could be interfer-
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ing in the Limulus Amoebocyte Lysate reaction, giving a false-positive result. These data deliver a cautionary note to laboratories who rely on the Limulus Amoebocyte Lysate test for endotoxin screening. If a new substance or system is used in the laboratory for which no enhancement or modifications in the Limulus Amoebocyte Lysate test are known, mouse embryo bioassays or rabbit pyrogen tests for endotoxins should be performed. Although the efficacy of the mouse embryo bioassay has been questioned (241, this is an instance in which it serves as the easiest and most relevant test available to an assisted reproduction technology laboratory. Low concentrations of Percoll and Nicodenz did not cause any adverse effects on mouse oocytes or sperm and there was no parthenogenetic activation. Adequate fertilization and development in vitro was obtained with both Percoll and Nicodenz containing medium. The in vivo development of these embryos was reduced compared with control embryos. Although adequate progression to the blastocyst stage was attained and the blastocysts had adequate cell numbers, there obviously was some interference with the embryos. The in vitro development of CD-l pronuclear embryos was unaffected by concentrations of Percoll and Nicodenz up to 20%. Development in 40% and 80% Percoll and Nicodenz was adequate fbr the first 48 hours of culture (72 hours after hCG1, at which point the embryos arrested. At this point, the embryos should begin to cavitate by the uptake of water, to form the blastocoel. The 40% and 80% Percoll and Nicodenz in the medium appeared to prevent the process of cavitation. When these embryos were moved to medium containing a greater percent of water, they were rescued to some extent. This arrest in development was more marked at higher concentrations of Percoll and Nicodenz and these embryos were less likely to be rescued when moved to new medium. It may be that 20% or 10% water in the medium is not sufficient for blastulation to occur. This also could explain the decreased implantation of the embryos grown in the systems tested (20% Percoll and Nicodenz). Shorter culture times or much lower concentrations may give viabilities more closely approximating the control groups. Unlike the effects of Percoll and Nicodenz, which left the embryos appearing healthy but suffering either developmental arrest or implantation failure, the washings from the Sperm Prep column caused the embryos to become atretic, suggesting a toxic effect rather than an interference with the developmental processes. Fertilization, survival, cell numbers, and viability all were compromised severely in
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the presence of the Sperm Prep Column washings. Although the nature of the toxic substance is not known, the way in which the embryos died in both the endotoxin tests and the embryo viability assays suggest the pathogenesis is not endotoxin related. Further, the washings showed a concentration-dependent decrease in both Limulus Amoebocyte Lysate reactivity and embryo toxicity. In conclusion, contrary to a former report (Svalander PC, Lundin K, Holmes PV, abstract), there was no evidence of endotoxin contamination in Perco11 or Nicodenz when using a mouse embryo bioassay as a test. Both products were co:mpatible with all aspects of mouse IVF, in vitro embryo development, and with some embryo viability. Further, human sperm had adequate survival in both Percoll and Nicodenz at low concentrations. Acknowledgment. The comments Ph.D., of Johns Hopkins University, Hygiene are gratefully acknowledged.
and editing of Alan Scott, School of F’ublic Health and
REFERENCES 1. Kruger TF, Acosta AA, Simmons RF, Swanson R-J, Matta JF, Oehninger S. Predictive value of abnormal sperm morphology in in vitro fertilization. Fertil Steril 1988;49:112-7. 2. Liu DY, Baker HWG. Acrosome status and morphology of human spermatozoa bound to the zona pellucida and oolemma determined using oocytes that failed to fertilize in vitro. Hum Reprod 1994;9:673-9 3. Van Steirteghem A, Nagy Z, Joris H, Liu J, Staessen C, Smitz J, et al. High fertilization and implantation rates after intracytoplasmic sperm injection. Hum Reprod 1983;8:1061-6. A, Tournaye H. Van der Elst J, Verheyen 4. Van Steirteghem G, Liebaers I, Devroey P. Intracytoplasmic sperm injection three years after the birth of the first ICSI child. Hum Reprod 1995; 10:2527-B. 5. Van der Zwalmen P, Lejeune B, Nijs M, Segal-Bertin G, Vandamme B, Schoysman R. Fertilization of an oocyte microinseminated with a spermatid in an in vitro fertilization programme. Hum Reprod 1995; 10:502-3. RJ, Clarkson JS. Cellular basis of defective sperm 6. Aitken function and its association with the genesis of reactive oxygen species by human spermatozoa. J Reprod Fertil 1987; 81:459-69. RJ, Clarkson JS, Fishel S. Generation of reactive oxy7. Aitken gen species, lipid peroxidation and human sperm function. Biol Reprod 1989;41:183-97. 8. Mortimer D. Sperm preparation techniques and iatrogenic failures of in vitro fertilization Hum Reprod 1991;6:173-6. JG, Lasso JL, Blasco L, Nunez C, Heyner S, Cabal9. Alvarez lero PP, et al. Centrifugation of human sperm induces sublethal damage; separation of human spermatcmzoa from seminal plasma by a dextran swim-up procedure without centrifugation extends their motile lifetime. Hum Repod 1993;8:108792. SJ, Fleming TP, Braude PR, Bohon VN, Gresham 10 Pickering GAG. Are human spermatozoa separated on a Percoll density gradient safe for therapeutic use? Fertil Sterill989; 51: 10249. 11 --. Fishel S, Jackson P, Webster J, Faratian B. Endotoxins in
Fertility
and
Sterilityl
12.
13.
14.
15.
16. 17.
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18. Whittingham DG. Parthenogenesis in mammals. In: Finn CA, editor. The Oxford reviews of reproductive biology. Oxford: Clarendon Press, 1980:205-31. 19. Cuthbertson KSR. Parthenogenetic activation of mouse oocytes in vitro with ethanol and benzyl alcohol. J Exp Zoo1 1983;226:311-4. 20. Cooper JF. Resolving Limulus Amoebocyte Lysate Test interferences. J Parenteral Sci Tech 1990;44:13-5. 21. Cooper JF. Ideal properties of a Limulus Amoebocyte Lysate reagent for pharmaceutical testing. Prog Clin Biol Res 1985; 189:241-g. 22. Twohy CW, Duran AP, Munson TE. Endotoxin contamination of parenteral drugs and radiopharmaceuticals as determined by the limulus amebocyte lysate method. J Parenteral Sci Tech 1984;38:190-201. 23. Arora M, Carver-Ward JA, Jaroudi KA, Sieck UV. Is Percoll safe for in vivo use? Fertil Steril 1994;61:97981. 24. Fleming TP, Pratt HPM, Braude PR. The use of mouse preimplantation embryos for quality control of culture reagents in human in vitro fertilization programs: a cautionary note. Fertil Steril 1987;47:858-60.
culture medium for human in vitro fertilization. Fertil Steril 1988;49:108-11. Scott LF, Sundaram SG, Smith S. The relevance and use of mouse embryo bioassays for quality control in an assisted reproductive technology program. Fertil Steril 1993;60:55968. Ord T, Patrizio P, Mare110 E, Balmaceda JP, Asch RH. MiniPercoll: a new method of semen preparation for IVF in severe male factor infertility. Hum Reprod 1990;5:987-9. Scott LA, Whittingham DG. The influence of genetic background and media components on the development of mouse embryos in vitro Mol Reprod Dev 1996;43:336-46. Quinn P, Wales RG. Growth and metabolism of preimplantation mouse embryos cultured in phosphate-buffered medium. J Reprod Fertil 1973;35:289-300. Whittingham DG. The culture of mouse ova. J Reprod Fertil Suppl 1971; 14:7-21. Montoro L, Subas E, Young P, Baccaro M, Swanson J, Sueldo C. Detection of endotoxin in human in vitro fertilization by the zona-free mouse embryo assay. Fertil Steril1990;54:10912.
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STERILIZE
Februaxy
Vol.67,No.2,
Mouse in vitro fertilization, embryo development and viability, human sperm motility in substances used for human sperm preparation for assisted reproduction* Lynette Samuel
199:
and
Scott, M.Sc.t Smith, M.D.
The Assisted Gy31ecology,
Reproduction Sin.ai Hospital,
Technologies Baltimore,
Program, Ma~~~~d
Women’s
Health
Center
at Sinai
Hospital,
L3epnrtmen.t
of Obstetrics
and
To investigate the endotoxin content and effects on mouse IVF and embryo develand human sperm motility of human sperm separation substances. De&u: One-cell and zona-free two-cell mouse embryo bioassays and Limulus Amoebocyte Lysate endotoxin tests and mouse oocyte parthenogenetic activation, IVF, preimplantation and postimplantation embryo development, and hnman sperm motility were performed in control medium or medium containing Percoll, Nicodenz, or the washings from Sperm Prep scphadcx columns. Setting: Research Laboratories, Sinai Hospital of Baltimore, Baltimore, Maryland. Main Outcome Measure(s): Endotoxin levels, embryo development, and sperm motility. Result(s): Mouse embryo bioassays indicated negative endotoxin levels in Percoll and Nicodenz but Limulus Amoebocyte Lysate assays had positive gel formation. Mouse IVF and preimplantation development was equivalent in control and Percoll- and Nicodenz-containing medium; none had parthenogenetic properties hut postimplantation development was reduced for embryos grown in the presence of Percoll or Nicodenz. Human sperm remained motile in the presence of Percoll or Nicodenz. Sperm Prep column washes were toxic to mouse gametes and embryos and human sperm and had gel formation with the Limulus Amoebocyte Lysate assay. Conclusion(s): Percoll and Nicodenz, but not Sperm Prep sephadex column washes, were compatible with mouse preimplalltation and postimplantation embryo development and human sperm motility. Fertil Sterile 1997;67:372-81 Objective:
opment
Key
Words:
Sperm
separating
substances,
Percoll,
The recovery of morphologically normal, intact, motile sperm from semen is required for use in most assisted reproductive techniques (1,2X The exception to this is direct intracytoplasmic sperm injection in which motile, nonmotile, immature, or even round sperm have been used for assisted fertilization (3-5).
Received April 25, 1996: revised and accepted July 29, 1996. * Presented in part at the 51st Annual Meeting of the American Society for Reproductive Medicine, Seattle, Washington, October 7 to 12, 1995. f Reprint requests: Lynette Scott, MSc.. Assisted Reproductive Technologies Program, Women’s Health Center at Sinai Hospital, 2411 West Belvedere Avenue, Weinberg Building, Suite 206, Baltimore, Maryland IFAX: ~10-578-8576~.
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There are a variety of sperm isolation protocols available to separate motile sperm froIm the remainder of the ejaculate. A simple method is direct swimup from the whole ejaculate. This method has proved to be inadequate in instances in which there is low density or motility because the efficiency of recovery is very low. Another widely used method has been double wash and swim-up in which the whole ejaculate initially is pelleted to remove the seminal fluid followed by swim-up from this pellet. However, centrifugation and pelleting of unselected sperm samples can have an adverse affect on sperm. This primarily is due to membrane damage caused by superoxide radicals produced by neutrophils and other cells that are pelleted during centrifugation
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(6-8). Repetitive centrifugation also mechanically can damage sperm, leading to decreased survival, motility, and function (9). An alternate approach is to separate the motile intact sperm from the rest of the ejaculate before pelleting. There are a number of ways to accomplish this, generally involving centrifugation on a density gradient made from polyvinylpyrrolidone-coated silica beads (Percoll, Nicodenz, or Ficoll) or migration through a sephadex matrix (Sperm Prep column) or glass wool. However, migration of the sperm through the separating substance can leave the sperm either coated with particles from the separation column (10) or mixed with some of the separating substance. Additional washing, and therefore centrifugation, of the sperm sample to eliminate these contaminants can induce damage (9). Furthermore, there have been reports of endotoxin contamination in certain of these products when using a Limulus Amoebocyte Lysate Test (Svalander PC, Lundin K, Holmes PV, abstract). Because endotoxins have been shown to be associated with increased fragmentation of human embryos (11) and nonviability of mouse embryos (12), it is thought that the sperm require washing, with centrifugation, to remove any traces of these substance before use in assisted fertilization techniques. In cases in which there is extremely low sperm density and/or motility, being able to use the separated sperm sample without additional washing and centrifugation would be beneficial for microdrop insemination (13) or assisted fertilization procedures (3, 4). Our laboratory uses the one-cell and zona-free two-cell mouse embryo bioassay for quality control (12). Using these tests and in other culture experiments there has been no evidence of endotoxin contamination in Percoll. Because previous reports of endotoxin contamination in Percoll used the Limulus Amoebocyte Lysate test, we screened Percoll for endotoxin content with both the mouse embryo bioassay and commercially available Limulus Amoebocyte Lysate tests. We further screened two other sperm separation substances, Nicodenz and Sperm Prep sephadex columns. The compatibility of the three separation substances with mouse IVF in vitro embryo development and embryo viability and human sperm motility in vitro also was studied as further bioassays. MATERIALS
AND
Sinai Hospital (Protocol Number Scott/Mou/l l-93). Mice were purchased from Charles River (Wilmington, VA). A colony of proven fertile CD-11males was maintained either for mating purposes or for sperm collection for IVF. Only fertile males with r6 months of successful mating were used for sperm collection for IVF. Virgin CD-l females at 4 to 5 weeks of age were superovulated by administration of 5 to 10 IU pregnant mare serum gonadotropin (G-4877; Sigma Chemical Company, St. Louis, MO) and chorionic gonadotrophin (CG-5) was given 44 to 48 hours apart. Females designated for mating .were placed individually with males overnight and checked the following morning for the presence of a vaginal plug as evidence of successful mating. A colony of vasectomized Fl (C57BV6NCr x C3W HeNCr) males were maintained for the induction of pseudopregnancy. Virgin CD-l females, 8 to 10 weeks old, were mated with the vasectomized males and checked for vaginal plugs. The day of plug was designated day 1 of pseudopregnancy. Media
METHODS
Mice
All protocols involving mice were approved by the Institutional Animal Care and Use Committee at
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All media were made from salts and tissue culture water (W-500) purchased from Sigma. The media used for all experiments were based on a modified Earle’s Balanced Salt Solution (EMO) containing no glucose or inorganic phosphate, with added ethylenediaminetetraacetic acid (0.11 mM) and glutamine (1.0 mM). The EM0 has been shown to support >80% fully expanded blastocyst development from the one-cell stage of a number of random and inbred strains of mice (12, 14). Variations of this medium containing either 0.5 mM glucose (EMG/0.5), 2.78 mM glucose (EMG), or 2.78 mM glucose and 1.32 mM inorganic phosphate (NaPOJ (EMGP) were also prepared (12,14). Basic medium (EMO) with 0.5 mM glucose (EMG/0.5) has been shown to give optimal development of CD-l embryos from the one-cell to blastocyst stage (Scott and Whittingham, unpublished data). All media were supplemented with 3 mg/mL bovine serum albumin (BSA, A-!3647; Sigma) unless otherwise stated. Media were prepared from concentrated stock solutions as required (12, 14); the osmolarity was adjusted to 280 to 290 mOsm, gassed with 5% COZ, 5% 02, 90% Nz for 5 minutes, sterilized with a 0.22pm filter, and placed in the incubator :for overnight equilibration. Solutions of 5%, lo%, 20%, 40%, and 80% Percoll (P-1644; Sigma) or Nicodenz (227510; Nicomet Pharma AS, Oslo, Norway) in EMG10.5 were formulated by substituting a percentage of the water content with undiluted Percoll or Nicodenz.
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Two lot numbers of Percoll and one of Nicodenz were used. Sperm Prep columns (ZLB Inc., Lexington, KY) were washed with 4 mL of EMG/O.5, which was collected in a sterile test tube. This initial 4 mL of media (SPCl) was the volume specified by the manufacturer as a prewash but was found to be highly toxic to mouse embryos. Thus, a series of washings were carried out in which four additional 2-mL washes were collected (SPCB, SPC3, SPC4, and SPC5), for a total of 12 mL of washing medium. For each of the three repeated experiments, three columns were used and the washings were pooled. There were two lot numbers of columns used. Embryo
Collection
and Culture
Superovulated, plugged CD-l females were killed at 20 hours after hCG. Pronuclear embryos were released from the oviducts directly into warmed EM0 containing 2 mg/mL hyaluronidase in order to remove the cumulus masses. The embryos were collected and washed through three 2-mL rinses of EM0 and two lOO-PL rinses of the medium in which they were to be cultured. Embryos were placed in groups of 10 per lo-p,L drop of medium under an oil overlay in a humidified, 5% COz in air incubator for culture. No HEPES buffer was used during oocyte or embryo manipulations to limit the number of environments the embryos were subjected to and to avoid intracellular pH changes that low bicarbonate can cause, which is known to reduce development in mouse and hamster embryos (15, 16). The number of embryos continuing to cleave and appear normal was recorded. An embryo was scored as surviving if it had at least two cells at 48 hours, four cells at 72 hours, eight cells at 96 hours, and was a blastocyst at 120 hours after hCG. All other embryos were scored as nonsurvivors as were those that were fragmenting. Endotoxin
Assays
Two commercially available endotoxin kits were used (BioWhittiker, Inc, Walkersville, MD; and TGF-1, Sigma). Both were based on a timed gel formation in the Limulus Amoebocyte Lysate test and were performed as per manufacturer’s instructions. Briefly, a concentration curve of both a known endotoxin and the sample were made and tested in the system. The test is based on the observation that as the levels of endotoxin increase the time for gel formation decreases and gel hardness increases when measured against the known incubation times for increasing concentrations of the standard. The last dilution of standard at which gel formation is
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observed after 1 hour of incubation is the positive endpoint. One-cell and zona-free two-cell mou.se embryo bioassays were performed (12, 17). In these tests the media were supplemented with 3 mg/mL polyvinyl alcohol (PVA, P-8136; Sigma) and not BSA. Embryos were collected as described in embryo collection and culture, except that two-cell embryos were flushed from the oviduct at 44 hours after hCG. These were washed through two 2 mL rinses of EMG/0.5 and then placed in acid-EMG/0.5 (EMG/0.5 made acidic, pH 3, by the addition of hydrochloric acid) for up to 5 seconds. They then were washed rapidly through four 2-mL rinses of EMG/0.5 before being placed in EMGi0.5 supplemented with 3 mg/mL PVA for culture. For all the endotoxin tests, a control sample of medium containing 1 ng/mL of Pseudomonas aeruginosa endotoxin (L7081; Sigma) and nlo BSA or PVA also was tested. The endotoxin content of all media used in the experiments (EMG/0.5 with and without BSA and PVA; EMG10.5 with added endotoxin; each of the sperm separation substances) was assessed using each of the endotoxin tests described above. Experiments were repeated three times for each system. Parthenogenetic
Activation
If oocytes are to be placed into any substance other than culture medium, the artificial activating properties of that substance should be known. Unfertilized mouse oocytes surrounded by the cumulus mass were released from the oviducts at 1.5 to 16 hours after hCG directly into the medium being tested. Test media were EMG/0.5 with 20%, 40%‘, or 80% Percoll or Nicodenz and SPCl, SPCS, SPC3, SPC4, and SPC5. They were incubated in the,se media for 6 hours and then released from their cumulus masses with hyaluronidase. Activation was carried out with the cumulus mass intact to simulate the conditions of human oocyte-cumulus complexes d.uring IVF. After the activation period, oocytes were scored for the extent of parthenogenetic activation (18, 19). Oocytes activated with 10% ethanol for 5 minutes or incubated in medium alone were used as activation controls (12, 19). Once scored, the oocytes were placed in culture and scored again the following day for any signs of activation and cleavage. Experiments were repeated three times with at least two cumulus masses per treatment in each repeat. In vitro
Fertilization
Superovulated females for oocyte collection were killed 12 to 14 hours after hCG and males were killed 24 hours before the IVF to allow the sperm to capac-
Fertility
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itate. After killing the male mice, the epididymis was excised and freed of as much of the connective tissue and blood vessels as possible, using sterile instruments. The contents of the epididymis were squeezed out, using sterile watchmakers forceps, into 2 mL EMGP supplemented with 10 mg/mL BSA and placed in the incubator at 37”C, with 5% CO2 in air. After 4 hours, the medium was aspirated gently out of the dish and the sperm concentration ascertained using a Makler counting chamber. The sperm were diluted with EMGP to approximately 2 x lo61 mL and then further diluted to 0.75 to 1.0 x lo6 motile sperm/ml with control medium or medium containing 40% Percoll, 40% Nicodenz, or the fourth washing from the Sperm Prep Column (SPC4). This resulted in approximately 20% Percoll and Nicodenz and half the amount of SP4 in the insemination medium. Drops of 100 I.~L of this sperm suspension were placed under oil as insemination drops. All oocyte and embryo manipulations were carried out under equilibrated mineral oil (M-3516; Sigma) on a heated microscope stage that was maintained at 36 to 37°C (12, 14). Oocyte-cumulus complexes intended for IVF were released from the oviduct into EMO, picked up with a flame-polished Pasteur pipette, and placed directly into the 100 /IL insemination drops. After 6 hours, 100 PL of EMG/0.5 with 2 mg/mL hyaluronidase (H-3506; Sigma) was added to the insemination drops to release the oocytes from their cumulus masses. The oocytes were washed with three 2-mL rinses of EMG/0.5 and then placed in lo-PL drops of this medium under oil. The oocytes were scored on an inverted microscope with Hoffman differential contrast optics at magnifications of X 3 10 and x500 for signs of fertilization, which were the presence of two polar bodies and two pronuclei. The number of oocytes fertilized in experimental media was compared with the number fertilized in control medium (EMGP). Fertilized oocytes were moved to EMG/0.5 for further development and grown for 100 hours (120 hours after hCG) as described above in embryo culture. The cell numbers in expanded blastocysts were obtained as described previously (14). Experiments were repeated three times with at least three cumulus masses per treatment group in each repeat. In Vitro
Embryo
tially, it was found that all embryos arrested at the morula stage when grown in 40% or 80% Percoll or Nicodenz. These embryos floated at the surface of the medium drops, at the oil-medium interface. If they were moved to 10% Percoll or Nicodenz after the initial 48 hours of culture, they continued growing. Thus, for the data presented for the 40% or 80% Percoll or Nicodenz, the embryos were grown for the first 48 hours in these concentrations and then moved to 10% Percoll or Nicodenz, respectively, for continued culture. All results were compared with control embryos cultured in EM0 with 0.5 mM glucose (EMG/0.5). Cell numbers in expanded blastocysts were obtained at 120 hours after hCG as described previously (15). Experiments were repeated three times with 20 embryos per group for each repeat. Embryo
The effect of exposing embryos to Percoll, Nicodenz, or SPC washes on their ability to implant and develop in pseudopregnant females was tested. For this, pronuclear embryos were cultured in 20% Per~011,20% Nicodenz, SPC4, or control medium for 72 or 88 hours. They were transferred to pseudopregnant females on the afternoon of day 3 of pseudopregnancy as morulae (96 hours after hCG) or blastocysts or on the morning of day 4 as expanded blastocysts (112 hours after hCG) (14). Elxperimental embryos were transferred to one uterine horn of pseudopregnant females and control embryos, grown in EMG/0.5, and transferred to the other. Five or six embryos at approximately the same developmental stage were transferred to each horn, thus making the choice of embryos for transfer nonrandom. This was to remove any implantation advantage any one group would have over another. However, the number of implantation sites and live pups on day 18 of pseudopregnancy were compared within a mouse (experimental versus control; paired t-test) and then overall between media (x2 analysis) for differences. Human
67, No. 2, February
Development
1997
Sperm
Preparation
and Motility
Sperm samples were collected by masturbation and allowed to liquefy for 30 minutes at 37°C. A basic semen analysis was performed (volume, density, motility). The sample then was separated on a two-step (40%, 80%) mini-Percoll (0.3 ImL) gradient (13). After centrifugation at 250 x g for 20 minutes, the upper layer of 40% Percoll and semen were removed. The sperm pellet then was aspirated off the bottom of the tube using a sterile Pasteur pipette. This pellet was placed in a centrifuge tube with care being taken not to touch the inside of the tube with
Pronuclear embryos were collected at 20 hours after hCG as described above and placed in different media for development over 96 hours (120 hours after hCG). The treatment groups were EMGj0.5, 5%, lo%, 20%, 40%, and 80% Percoll and Nicodenz and each of the Sperm Prep column washings. Ini-
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the outside of the pipette. The sperm pellet was suspended in 2 mL basic medium with 2.78 mM glucose (EMG) and aliquoted into eight 0.4-mL volumes. The aliquots were resuspended in 2 mL of EMG containing 20% or 40% Percoll, 20% or 40% Nicodenz, and 100% of each of SPCl, SPC3, SPC5, or control medium. They were centrifuged for 5 minutes at 250 x g and the final pellet was resuspended in 0.5 mL of the medium to which they were designated. The percent of motile sperm was then assessed after 8 and 24 hours of incubation at 37°C and compared with the motility in the control medium. The data were analyzed between media within a sample (t-test and analysis of variance [ANOVAI) and overall between the media. RESULTS Endotoxin
Contamination
The CD-l embryos showed no signs of fragmentation in either a one-cell or zona-free two-cell mouse embryo bioassay when cultured in control medium or medium with 20% Percoll or 20% Nicodenz (Fig. 1). In all these treatments there was >70% expanded blastocyst formation by 120 hours after hCG, which compares favorably with results previously reported for CD-l embryos in EMG (12, 14). In contrast, mouse embryo development from the one-cell and two-cell stage in SPC washes was impaired severely. In both bioassay systems, all embryos became atretic within an hour of being placed in SPCl, SPCB, or SPC3. There was limited development and
survival in the SPC4 and SPC5. Thfe embryos became dark and atretic but did not display any fragmentation and membrane perturbations that are seen with endotoxin contamination and in endotoxin-spiked medium. Control medium containing 1 ng/mL of endotoxin resulted in 0% blastocyst formation, cell membrane perturbations, extreme fragmentation, and developmental arrest, Using the Limulus Amoebocyte Lysate kits, control medium (EMG/0.5) supplemented with BSA or PVA was negative for endotoxin. Control medium spiked with 1 ng/mL endotoxin resulted in gel formation in both test kits at a level indicating approximately 1 ng/mL endotoxin (data not shown). The Limulus Amoebocyte Lysate kits worked consistently well with concentration curves of endotoxin and on other products. However, both Percoll and Nicodenz had gel formation in both Limulus Amoebocyte Lysate kits. The time of gel formation and the extent of gelling was extremely variable, dependent on the concentration of either substance and the buffer used as diluent (data not shown). Sperm Prep column washings had gel form.ation on both test kits. The extent of gelling decreased with washing number. Parthenogenetic
Normally aging untreated oocytes spontaneously activate at very low levels in vitro (5%; 71147 oocytes from seven cumulus masses) (data not shown). Control ethanol activations resulted in 58% activation (88/152 oocytes from eight cumulus masses) with 49% (43/88) of activated oocytes cleaving to at least the eight-cell stage. The level of activation in 20% Percoll(6%; 8/134, six cumulus masses’),40% Percoll (3%; 4/123, five cumulus masses), 80% Percoll (4%; 5/119, five cumulus masses), and 20% Nicodenz (5%; 5/107, four cumulus masses), 40% Nicodenz (1%; l/112, five cumulus masses), and 80% Nicodenz (7%; g/126, six cumulus masses) was not significantly greater than that obtained with aging untreated oocytes. Sperm Prep column washings SPCl, SPCB, and SPC3 did not activate oocytes; they caused the oocytes to become dark and atretic in appearance whereas SPC4 and SPC5 resulted in no activation and minimal atresia in the oocytes (data not shown). In Vitro
Figure 1 Data represent the mean 2 SEM of three replicate mouse embryo bioassays for endotoxin using the one-cell and zona-free two-cell tests. All media systems were tested in each repeat experiment.
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Activation
Fertilization
There were no differences in the percentages of oocytes fertilized in control medium or medium containing 20% Percoll or 20% Nicodenz (Table 1). The exposure of sperm and oocytes to a 1 :l dilution of SPC4 resulted in a decrease in the number of oocytes
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Table 1 IVF of CD-l Oocytes With Sperm Prepared With of the Resulting Embryos for 100 Hours in Embryo Culture
Control Medium
(EMGP) and Experimental (EMGIO.5)” Medium
Control No. No. No. No. Cell
of cumulus masses of oocytes of fertilized oocytes$ of blastocytes$ no. in blastocystsl
medium?
Table 2 The In Embryos Grown Pronuclear Stage (120 Hours After
1997
9 iii (49 9.3)s 29 (60: 4.7)1/ 42 (8.4)*”
Vitro Development and Cell in Control and Experimental (20 Hours After hCG) to the hCG) From Three Repeated
Number of CD-l Media From the Blastocyst Stage Experiments
Blastocyst developmentt
Culture system”
Development
67, No. 2, February
10 108 68 (63, 6.2) 51 (75, 4.1) 60 (3.9)
There was no survival of CD-l embryos in the first three SPC washes. The SPC4 and SPC5 resulted in better survival but this was significantly less than controls (P < 0.001). The embryos in t.hese experiments became atretic, fragmented, and did not
There were no differences in blastocyst development of CD-1 mouse embryos grown in control medium or with 5%, lo%, or 20% Percoll or Nicodenz (Table 2). Higher concentrations of Percoll and Nicodenz resulted in a slight decrease in blastocyst formation (P < 0.05). Development of embryos in 80% Nicodenz for the first 48 hours was reduced significantly compared with development in lower concentrations of Nicodenz or Percoll or in control medium (P = 0.001, paired t-test and ANOVA). The cell numbers in control embryos and in those cultured in 5%, lO%, and 20% Percoll or 5% or 10% Nicodenz were not significantly different. There was a reduction in the cell numbers of embryos grown in 20% Nicodenz (P < 0.05). There was a reduction in cell number of embryos initially grown in 40% Perco11(P < 0.05) or 40% Nicodenz (P < 0.01) and of those initially grown in 80% Percoll (P < 0.001) or 80% Nicodenz (P < 0.001) compared with control medium.
Vol.
0.5 x SPC4’F
0 P < 0.0005 versus control. 11P < 0.005 versus control. n Values in parentheses are SEM. **P < 0.001 versus control,
fertilized (P < 0.005, t-test and ANOVA). The mouse sperm in these insemination drops had decreased motility and survival compared with that seen in control medium, Percoll-, and Nicodenz-containing medium. The development to blastocysts was equivalent for embryos fertilized in insemination medium made from control, Percoll, and Nicodenz but was reduced for embryos resulting from 0.5 x SPC4 insemination drops (P < 0.005). The cell numbers in expanded blastocysts after 100 hours of culture followed a similar pattern. There were no differences in the cell numbers of embryos resulting from insemination in control, Percoll, or Nicodenz insemination media. There was a significant reduction in cell number in embryos resulting from insemination in a 1:l dilution of SPC4 insemination medium (P < 0.001). Embryo
20% Nicodenzt
12 114 74 (65, 4.7) 52 (70, 1.91 58 (2.7)
* Data are the sum of three repeat experiments, t All IVF was in EMGP and subsequent embryo culture was in EMGI0.5. f Values in parentheses are percentages and SEM, respectively.
and the Devel,opment
system
20% Percollt
9 97 65 (67, 3.4) 47 (72, 2.8) 61 (1.8)
Medium
Scott
EMGI0.5 Percoll 5% 107c 20% 40% 80% Nicodenz 5% 10% 20% 40% 80% Sperm prep SPC-1 SPC-2 SPC-3 SPC-4 SPC-5
Total embryos cultured
180
Blastocyst cell no.+
Mean ? SEM
Tota I embryos counted
Mean ? SEM
83 5
2.7
30
66 -t 2.3
60 60 60 60 60
72 75 77 63 62
+ t 2 + i
2.5 2.3 2.9 2.98 2.91,
30 30 30 30 30
65 67 64 56 43
k 2 + k t
1.9 1.5 1.5 1.69 2.1(1
60 60 60 60 60
77 69 69 63 35
+ 3.4 2 5.1 2 4.3 ? 10.95 5 5.61
30 30 30 30 30
66 58 56 47 34
I i -+ + 5
2.2 2.1 1.85 1.911 2.31
0 0
-
37 2 2.3$ 44 -t 2.517
60 60 60 60 60
5 x 40 2 52 2
1.g**: 7.11 3.01
14 20
* Culture system in which the embryos were grown. t Percent blastocyst and expanded blastocyst development at 120 hours after hCG. $ Cell number in blastocysts and expanded blastocysts at 120 hours after hCG. 5 P < 0.05 versus controls. I/P < 0.01 versus controls. ll P < 0.001 versus controls. **p < 0.0001.
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Table 3 The Implantation and Fetal Development of CD-l Embryos at 18 Days of Gestation That Were Cultured (Control) or in EMG/0.5 Containing 20% Percoll, 20% Nicodenz, or SPC4 (Experimental) for 76 or 92 Hours (96 and 112 Hours After hCG) 20% Control No. of pregnant recipients No. of embryos transferred No. of embryos implanted No. of live fetuses Implantation* (%) Live$ (%‘o)
Percoll
* Percent of transferred embryos tP < 0.05 versus EMGI0.5. $ Percent of transferred embryos
20% Nicodenz
Experimental
6 33 26 18 79 55
Control
6 33 22 16
Control
6 33 18 3 551 3%
3 P < 0.01 versus 11P < 0.001 versus
4 17 11 10 65 59
4 15 3 2
2Oll 1311
EMG/0.5. EMG/O.S
hours resulted in a decrease in motility compared with control medium (P < 0.05). After 24 hours, the motility was reduced further for both Percoll (P < 0.01) and Nicodenz (P < 0.001).
Viability
There was no difference in the implantation rates of morulae or blastocysts grown in 20% Percoll or control medium (Table 3). There was a slight but significant decrease in the number of live fetuses seen with embryos grown in 20% Percoll. Both the implantation (P < 0.05) and the number of live fetuses (P < 0.01) were reduced when embryos were grown to the morula and blastocyst stage in Nicodenz compared with those grown in control medium. There was very poor implantation and fetal development of morulae and blastocysts grown in SPC4 compared with control medium and Percoll and Nicodenz cultures.
8 Hours
I *
*** ai**
EMG
20%
40%
PWCOII
Human Sperm Substances
Motility
in Sperm
Separation
The motility of the sperm samples at 8 and 24 hours of incubation in control (EMG) and experimental media are shown in Figure 2. In control medium, there was a decrease in the percent of motile sperm with time that was sample dependent. Some individual samples had a significant reduction in motility after both 8 and 24 hours of incubation. A similar pattern was seen for each individual sample in both Percoll and Nicodenz, thus the data is presented as the mean + SEM survival. Incubation in 20% Percoll or 20% Nicodenz for 8 hours did not significantly alter the motility of the sperm samples when compared with the same samples in test medium. However, after 24 hours incubation, there was a reduction in motility in both 20% Percoll (P < 0.01) and 20% Nicodenz (P < 0.001) compared with control medium. Incubation in 40% Percoll or 40% Nicodenz for 8
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Experimental
in a live fetus.
cleave. The cell numbers in embryos grown in SPC4 and SPC5 were significantly less than those grown in control medium (P < 0.001). Embryo
SPC-4
Experimental
6 34 23 17 68 50
implanting. resulting
in EMG/0.5
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loo 75
hi !O%
40%
SFCI
SC3
WCS
NlCCKh2
24 Hours
-Al
0 EMG
20%
e
20%
40%
WC1
SK3
spcs
Figure 2 Data represent the mean -t SEM motility at 8 and 24 hours of 10 samples in control (EMG) and experimental media. Each sample acted as its own control. Patterns of motility were the same for each sample in the different media, although the samples were different from each other. The data were analyzed within a sample and then overall for media differences. Control medium was EMG, which was the diluent for Percoll, Nicodenz, and SPC washes. *P < 0.05; **P < 0.01; ***P ,< 0.001.
Fertility
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Sperm motility in SPCl after 8 hours incubation was decreased significantly compared with control medium, Percoll, and Nicodenz (P < 0.0001). After 24 hours incubation, there were no motile sperm in SPCl. Sperm motility in SPC3 was slightly better but still significantly less than that obtained with control medium, Percoll, and Nicodenz at both 8 and 24 hours of incubation (P < 0.001). The final washing, SPC5, resulted in sperm motility that was reduced at 8 hours of incubation (P < 0.05) and 24 hours of incubation (P < 0.0001). However, these data were no different to those obtained with 40% Percoll and Nicodenz at 24 hours and 20% and 40% Nicodenz at 24 hours of incubation. DISCUSSION
The results presented here indicate that polyvinylpyrrolidone-coated silica bead density gradient sperm separation substances such as Percoll and Nicodenz are compatible with mouse oocytes IVF and embryo development. However, there was reduced viability of embryos grown for extended periods in these substances at a concentration of 20%. Human sperm remained motile in 20% Percoll and Nicodenz for 24 hours. In contrast, the wash medium from a Sephadex-based sperm separation column was highly toxic to mouse oocytes, embryos, and human sperm. The toxic substances washed off the columns appeared to decrease with multiple washes but a significant cytotoxic effect was still noted after 12 mL of wash, which was equivalent to six column volumes of wash. The presence of endotoxins in embryo culture media and on other embryo contact materials is harmful to embryos. Mouse (12, 17) and human embryos (11) both exhibit increased fragmentation and reduced viability in the presence of endotoxins. In this study, endotoxin contamination was not found in either Percoll or Nicodenz using the mouse embryo bioassay. It has been reported that Percoll has high concentrations of endotoxins (Svalander PC, Lundin K, Holmes PV, abstract) when tested with a Limulus Amoebocyte assay. In these experiments, gel formation, which is consistent with endotoxin contamination, was found with in both of Percoll and Nicodenz when using a Limulus Lysate Assay. Because there was no evidence of endotoxins with the mouse embryo bioassay, it was speculated that this gel formation was an artifact. The use of the Limulus Amoebocyte Lysate assay to test for endotoxin contamination in raw products has been reported to be problematic due to poor specificity. It initially was found that only a small percentage of substances initially screened could be
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tested without modifications. This was due to either inhibitory or enhancement mechanisms, which resulted in false-positive or -negative tests (20-22). There are many conditions that can affect the Limulus Amoebocyte Lysate reaction, which is a cascade of serine protease reactions triggered by endotoxin and finally resulting in the formation of a gel. The reaction is sensitive to pH; the presence 1orabsence of divalent cations; the presence or absenoe of cellulose materials that contain Limulus Amoebocyte Lysatereactive material, typically B-glycans; oxidants or antioxidants that can affect the protease reactions; and serum proteolytic enzymes. The reaction mix has slight buffering capacity between pH 6 and pH 8. Enhancement of the reaction can occur if the concentrations of either or both Mg2+ and Ca2+ are not optimized. When tested in the rabbit pyrogen test, most Limulus Amoebocyte Lysate-reactive material is negative and does not cause any other in vitro effects. Percoll and Nicodenz are colloidal suspensions of silica coated with polyvinylpyrrolidone. They have a room temperature pH of 9.0. Both will spontaneously gel as the pH is lowered. It was found that simply lowering the pH of Percoll to 6.6 and Nicodenz to 6.0 resulted in spontaneous gelling (data not shown). Certain buffer systems also caused gelling of Percoll and Nicodenz. The gelling effect was augmented by an increase in temperature and with increased concentrations of Percoll and Nicodenz. Further, when 100% Percoll was mixed with the Limulus Amoebocyte Lysate reaction mix (1:5 ratio), the resulting pH was 6.8. If a bicarbonate (Earle’s blalanced salt solution) or a phosphate buffer initially was used to dilute the Percoll to 50%, the pH was decreased to 6.2. In both instances, there was gel formation within seconds of adding the Limulus Amoebocyte Lysate reaction ingredients to the tube. These data suggest that the positive Limulus Amoebocyte Lysate endotoxin test seen with Percoll and Nicodenz could in fact be false positives related to the combined effects of the properties of both the Limulus Amoebocyte Lysate test and the nature of Percoll and Nicodenz. The absence of i:nflammatory and other cellular responses when Percoll was injected directly into the uterine horns or ovaries of rabbits supports this contention (23). The toxicity of the wash medium from the Sperm Prep columns on mouse embryos and the positive reaction on the Limulus Amoebocyte Lysate tests made it hard to draw conclusions as to the endotoxin content of these columns. The mouse embryo toxicity could be due to any preservative, buffer, or fungicide that may be in the Sephadex to preserve the columns before use, These substances also could be interfer-
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ing in the Limulus Amoebocyte Lysate reaction, giving a false-positive result. These data deliver a cautionary note to laboratories who rely on the Limulus Amoebocyte Lysate test for endotoxin screening. If a new substance or system is used in the laboratory for which no enhancement or modifications in the Limulus Amoebocyte Lysate test are known, mouse embryo bioassays or rabbit pyrogen tests for endotoxins should be performed. Although the efficacy of the mouse embryo bioassay has been questioned (241, this is an instance in which it serves as the easiest and most relevant test available to an assisted reproduction technology laboratory. Low concentrations of Percoll and Nicodenz did not cause any adverse effects on mouse oocytes or sperm and there was no parthenogenetic activation. Adequate fertilization and development in vitro was obtained with both Percoll and Nicodenz containing medium. The in vivo development of these embryos was reduced compared with control embryos. Although adequate progression to the blastocyst stage was attained and the blastocysts had adequate cell numbers, there obviously was some interference with the embryos. The in vitro development of CD-l pronuclear embryos was unaffected by concentrations of Percoll and Nicodenz up to 20%. Development in 40% and 80% Percoll and Nicodenz was adequate fbr the first 48 hours of culture (72 hours after hCG1, at which point the embryos arrested. At this point, the embryos should begin to cavitate by the uptake of water, to form the blastocoel. The 40% and 80% Percoll and Nicodenz in the medium appeared to prevent the process of cavitation. When these embryos were moved to medium containing a greater percent of water, they were rescued to some extent. This arrest in development was more marked at higher concentrations of Percoll and Nicodenz and these embryos were less likely to be rescued when moved to new medium. It may be that 20% or 10% water in the medium is not sufficient for blastulation to occur. This also could explain the decreased implantation of the embryos grown in the systems tested (20% Percoll and Nicodenz). Shorter culture times or much lower concentrations may give viabilities more closely approximating the control groups. Unlike the effects of Percoll and Nicodenz, which left the embryos appearing healthy but suffering either developmental arrest or implantation failure, the washings from the Sperm Prep column caused the embryos to become atretic, suggesting a toxic effect rather than an interference with the developmental processes. Fertilization, survival, cell numbers, and viability all were compromised severely in
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the presence of the Sperm Prep Column washings. Although the nature of the toxic substance is not known, the way in which the embryos died in both the endotoxin tests and the embryo viability assays suggest the pathogenesis is not endotoxin related. Further, the washings showed a concentration-dependent decrease in both Limulus Amoebocyte Lysate reactivity and embryo toxicity. In conclusion, contrary to a former report (Svalander PC, Lundin K, Holmes PV, abstract), there was no evidence of endotoxin contamination in Perco11 or Nicodenz when using a mouse embryo bioassay as a test. Both products were co:mpatible with all aspects of mouse IVF, in vitro embryo development, and with some embryo viability. Further, human sperm had adequate survival in both Percoll and Nicodenz at low concentrations. Acknowledgment. The comments Ph.D., of Johns Hopkins University, Hygiene are gratefully acknowledged.
and editing of Alan Scott, School of F’ublic Health and
REFERENCES 1. Kruger TF, Acosta AA, Simmons RF, Swanson R-J, Matta JF, Oehninger S. Predictive value of abnormal sperm morphology in in vitro fertilization. Fertil Steril 1988;49:112-7. 2. Liu DY, Baker HWG. Acrosome status and morphology of human spermatozoa bound to the zona pellucida and oolemma determined using oocytes that failed to fertilize in vitro. Hum Reprod 1994;9:673-9 3. Van Steirteghem A, Nagy Z, Joris H, Liu J, Staessen C, Smitz J, et al. High fertilization and implantation rates after intracytoplasmic sperm injection. Hum Reprod 1983;8:1061-6. A, Tournaye H. Van der Elst J, Verheyen 4. Van Steirteghem G, Liebaers I, Devroey P. Intracytoplasmic sperm injection three years after the birth of the first ICSI child. Hum Reprod 1995; 10:2527-B. 5. Van der Zwalmen P, Lejeune B, Nijs M, Segal-Bertin G, Vandamme B, Schoysman R. Fertilization of an oocyte microinseminated with a spermatid in an in vitro fertilization programme. Hum Reprod 1995; 10:502-3. RJ, Clarkson JS. Cellular basis of defective sperm 6. Aitken function and its association with the genesis of reactive oxygen species by human spermatozoa. J Reprod Fertil 1987; 81:459-69. RJ, Clarkson JS, Fishel S. Generation of reactive oxy7. Aitken gen species, lipid peroxidation and human sperm function. Biol Reprod 1989;41:183-97. 8. Mortimer D. Sperm preparation techniques and iatrogenic failures of in vitro fertilization Hum Reprod 1991;6:173-6. JG, Lasso JL, Blasco L, Nunez C, Heyner S, Cabal9. Alvarez lero PP, et al. Centrifugation of human sperm induces sublethal damage; separation of human spermatcmzoa from seminal plasma by a dextran swim-up procedure without centrifugation extends their motile lifetime. Hum Repod 1993;8:108792. SJ, Fleming TP, Braude PR, Bohon VN, Gresham 10 Pickering GAG. Are human spermatozoa separated on a Percoll density gradient safe for therapeutic use? Fertil Sterill989; 51: 10249. 11 --. Fishel S, Jackson P, Webster J, Faratian B. Endotoxins in
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18. Whittingham DG. Parthenogenesis in mammals. In: Finn CA, editor. The Oxford reviews of reproductive biology. Oxford: Clarendon Press, 1980:205-31. 19. Cuthbertson KSR. Parthenogenetic activation of mouse oocytes in vitro with ethanol and benzyl alcohol. J Exp Zoo1 1983;226:311-4. 20. Cooper JF. Resolving Limulus Amoebocyte Lysate Test interferences. J Parenteral Sci Tech 1990;44:13-5. 21. Cooper JF. Ideal properties of a Limulus Amoebocyte Lysate reagent for pharmaceutical testing. Prog Clin Biol Res 1985; 189:241-g. 22. Twohy CW, Duran AP, Munson TE. Endotoxin contamination of parenteral drugs and radiopharmaceuticals as determined by the limulus amebocyte lysate method. J Parenteral Sci Tech 1984;38:190-201. 23. Arora M, Carver-Ward JA, Jaroudi KA, Sieck UV. Is Percoll safe for in vivo use? Fertil Steril 1994;61:97981. 24. Fleming TP, Pratt HPM, Braude PR. The use of mouse preimplantation embryos for quality control of culture reagents in human in vitro fertilization programs: a cautionary note. Fertil Steril 1987;47:858-60.
culture medium for human in vitro fertilization. Fertil Steril 1988;49:108-11. Scott LF, Sundaram SG, Smith S. The relevance and use of mouse embryo bioassays for quality control in an assisted reproductive technology program. Fertil Steril 1993;60:55968. Ord T, Patrizio P, Mare110 E, Balmaceda JP, Asch RH. MiniPercoll: a new method of semen preparation for IVF in severe male factor infertility. Hum Reprod 1990;5:987-9. Scott LA, Whittingham DG. The influence of genetic background and media components on the development of mouse embryos in vitro Mol Reprod Dev 1996;43:336-46. Quinn P, Wales RG. Growth and metabolism of preimplantation mouse embryos cultured in phosphate-buffered medium. J Reprod Fertil 1973;35:289-300. Whittingham DG. The culture of mouse ova. J Reprod Fertil Suppl 1971; 14:7-21. Montoro L, Subas E, Young P, Baccaro M, Swanson J, Sueldo C. Detection of endotoxin in human in vitro fertilization by the zona-free mouse embryo assay. Fertil Steril1990;54:10912.
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