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Evaluation of human serum as a culture medium for studies of rat embryonic development in vitro
Reproductive Toxicology, Vol. 2, pp. 135-140, 1988
0890-6238/88$3.00 + .00 Copyright (~ 1988PergamonPress pie
Printed in the U.S.A.
EVALUATION
OF HUMAN SERUM OF RAT EMBRYONIC
AS A CULTURE DEVELOPMENT
MEDIUM FOR STUDIES IN VITRO
MOHAMMAD ANWAR* and FELIX BECKt Departments of ?Anatomy and *Obstetrics & Gynaecology, University of Leicester, University Road, Leicester, England Abstract m The normal growth of rat embryos cultured in human serum supplemented with glucose and 10% rat serum is of potential use in the assessment of teratogenic risk in man. Before this technique can be widely applied, it is necessary to know whether the stages of the menstrual cycle at which a serum sample is tested materially affect the results obtained. Moreover, in order to achieve reproducible conditions we have found it necessary to add a minimal amount of rat serum to the human serum used for culture, but unnecessarily high levels of rat serum supplementation could over-compensate for any growth factor deficiency in human serum. Here we report that culture of rat conceptuses gives similar results irrespective of whether human first, second, or third trimester pregnancy serum, postnatal serum, or serum at various stages of the menstrual cycle is used. We also report that addition of 2% rat serum supplement is sufficient to achieve reproducible rat embryonic growth and differentiation in human serum. Key Words: Teratology, Whole e m b r y o culture, In vitro drug evaluation.
INTRODUCTION
embryopathic properties of serum obtained from epileptic patients on various anticonvulsants. A review of the original data reported by Chatot et al. (3) shows that 20% of the embryos cultured in normal human serum developed abnormally. This makes it difficult to use the original method in critical experiments because no acceptable " n o r m a l " baseline for controls is available. In an attempt to achieve reproducible rat embryonic growth and differentiation, Reti et al. (5) diluted human serum with 10% rat serum and supplemented glucose to a final concentration o f 3 mg/mL. They reported that embryonic growth and differentation as measured by protein levels and morphological assessment was comparable with that occurring in pure rat serum and also that the embryos had significantly fewer developmental anomalies when compared with those grown in glucose-only supplemented human serum. L e a r et al. (6) and Gupta and Beck (7), using the same conditions, also reported a significant improvement in morphological scores, hemoglobin contents, and protein values, with a lower range o f abnormalities when rat conceptuses were cultured in human serum that had been supplemented with glucose and 10% rat serum. These authors proposed that a small aliquot of rat serum added to the human serum provides only the species-specific growth factors required by the rat embryos to differentiate normally. In support of
In 1970 Tanimura and Shepard (1) reported that N e w ' s (2) roller bottle in-vitro embryo culture technique could be used to grow postimplantation rat conceptuses in human serum. Since then many workers, including Chatot et al. (3), Porov et al. (4), Reti et al. (5), L e a r et al. (6), Gupta et al. (7), Priscott (8), and Steele (9), have performed in-vitro e m b r y o culture experiments in undiluted human serum and have published inconsistent results. The inconsistencies within the reported results include unacceptably high levels of developmental anomalies as well as a varying protein content among cultured embryos. C h a t o t et al. (3) suggested that postimplantation rat conceptuses developed more successfully when undiluted human serum was supplemented with glucose to a final concentration of 3 mg/mL. Glucose supplementation was thought to be necessary, as the glucose level in rat serum (1.8 mg/mL) is higher than the human serum level (0.8 mg/mL). These authors proposed that rat whole e m b r y o culture could be used in a clinical context to screen for embryopathic factors in human serum. A year later Chatot et al. (10) applied this method to test the A d d r e s s c o r r e s p o n d e n c e to: Dr. M. A n w a r , D e p a r t m e n t of Obstetrics & Gynaecology, Leicester Royal Infirmary, Leicester, England. Received 16 February 1988; Accepted 4 July 1988. 135
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this hypothesis, A1-Alousi (11) has shown that 10% rat serum diluted with 90% Cockroft's (12) balanced salt solution and appropriately supplemented with glucose and vitamins was insufficient to support embryonic growth. From this it may be inferred that the growth factors in the rat serum (lacking in human serum) are required for the embryos to grow and differentiate to their full capacity in human serum. Beck et al. (13) used this modified technique to demonstrate the teratogenic properties of human serum obtained from volunteers who had consumed alcohol under experimental conditions. It is clear that if the in-vitro embryo culture technique is to be applied in a clinical context the conditions under which the experiments are performed must be standardized. The maternal "milieu interieur" during the menstrual cycle and pregnancy is altered to create conditions favorable for implantation and development. Apart from a changing hormonal profile, there are widespread changes in the constituents of serum during pregnancy, including a fall in serum proteins, pH, urea, and electrolytes, consequently leading to reduced osmolality as discussed by Hytten and Leitch (15). These changes could alter the embryo growthsupporting capacity of human serum. It is thus important to define the stage of the menstrual cycle and of gestation at which the serum is potentially suitable for in-vitro culture. Although Buckley et al. (16) has reported that equally satisfactory results were obtained when serum from male, nonpregnant, and pregnant rats was used as a culture medium, no comparable studies have been performed using serum obtained from humans. Furthermore, if human serum is supplemented with rat serum, there is no consensus regarding the optimum amount of rat serum required for standard embryonic growth and differentiation. If biologically and clinically meaningful conclusions are to be drawn from the culture of rat embryos in human serum, it is important that supplementation with rat serum is kept to a minimum. The addition of unnecessarily high levels of rat serum makes it possible that deficiencies present in the human serum could be compensated by the added rat serum. MATERIALS AND METHODS
Selection of patients and the preparation of serum Blood was obtained from female medical students, nursing staff, patients attending antenatal clinics, and mothers in the postnatal wards between the hours of 0900 and 1000, after their normal breakfasts. Apart from patients on hematinic supple-
Volume 2, Number 2, 1988
mentation, those volunteers on oral contraceptives or any other drug therapy were excluded. Blood samples were obtained from women who, according to their last normal menstrual period, were in: 1) The first half of the menstrual cycle (day 5 to 14) 2) The second half of the menstrual cycle (day 15 to 28) 3) The first trimester of pregnancy (up to 14th gestational week) 4) The second trimester of pregnancy (15th to 28th gestational week) 5) The third trimester of pregnancy (29th to 42nd gestational week) 6) The post-delivery period (up to 5 days) For experiments to study the effects of supplementing human serum with various concentrations of rat serum, blood samples were obtained only from women in the second half of their menstrual cycle. Thirty mL human blood samples were centrifuged immediately after venesection and treated according to the method of New (17). Prior to use, human serum was always supplemented with glucose to a final concentration of 3 mg/mL and with an appropriate amount of rat serum; 0.02 mL of antibiotics (stock solution containing streptomycin 5000/zg/mL and penicillin 5000 Iu/mL) were added per mL of all culture media. Cultures were gassed initially with a mixture of 5% 02, 5% CO2, and 90% N2. After 24 hours this was replaced with 20% 02, 5% CO2 and 75% Nz and after 42 hours with 40% 02, 5% CO2 and 55% N2. For control experiments, conceptuses were cultured in pooled rat serum obtained from several pregnant animals.
Explantation of rat conceptuses Conceptuses were obtained from Wistar rats at a later time of 1100 hours of the 10th day of gestation (timed from midnight preceding the morning on which vaginal plugs were observed). They were removed under aseptic conditions in Hank's balanced salt solution and, in order to standardize the experiment, only those at the late head-fold stage of development (3 somites) were selected for the culture experiments. Embryo culture Conceptuses from each litter were randomly divided into experimental and control groups. Embryos were cultured at 37 °C for 48 hours in rotating
137
Rat embryo culture in human serum • M. ANWARand F. BECK Table 1. Embryonic growth in pure and supplemented (10% rat serum) human serum 10% rat serum supplementation Growth parameter
invivo
100% rat serum
100% Postovul hum serum
(A) Preovul
(B) Postovul
7
10
10
50
110
110
(D) 2ndtrim
(E) 3rdtrim
(F) Postnatal
9
10
9
9
90
95
90
90
42.56 0.13
42.66 0.14
42.75 0.11
42.45 0.19
(C) lsttrim
No. of patients No. of embryos
25
Morphological score S.E.
42.96 0,03
42.64 0.08
40.26 0.68**
3,94 0.06
3.90 0.04
3.76 0.05**
4.14 0.03**
4.25 0.03**
4.16 0.04
4.28 0.02**
4.25 0.04**
4.00 0.04**
3.33 0.05
3.33 0.04
2.96 0.05**
3.42 0.02**
3.42 0.03*
3.43 0.04
3.49 0.02**
3.43 0.03
3.26 0.03
26.86 0,09
26.50 0.09
24.94 0.16"*
289.91 13.50
215.02 4.15"*
132.58 3.51"*
Yolk sac diameter (mm) S.E. Crown-rump length (ram) S.E. Somite number S.E. Protein (/xg) S.E. Abnormal embryos
(Xz)
0
106
3
l0 (5.48**)
42.55 0.07
42.55 0.08
26.32 0.09
26.37 0.13
26.36 0.14
26.48 0.10
25.94 0.10
209.29 4.08
212.59 4.99
203.78 5.67
205.59 4.13
201.99 5.33
203.40 4.67
4 (0.1)
3 (0.01)
3 (0.04)
3 (0.02)
4 (0.35)
26.12 0.12"
4 (0.35)
ovul = ovulatory; trim -- trimester; No. = number of embryos. *p < 0.02 when compared with embryonic growth in whole rat serum. **p < 0.01 when compared with embryonic growth in whole rat serum.
glass bottles according to the method o f N e w (17). E a c h bottle contained 5 conceptuses in 5 m L of the culture medium. On average, 10 conceptuses per human serum sample were cultured. H u m a n serum was supplemented with glucose and various concentrations of rat serum. Control experiments were performed in 100% rat serum.
Assessment of embryonic growth and development At the end o f the culture period conceptuses were examined under a dissecting microscope. As a measure o f development their maximum yolk sac diameters and crown/rump lengths were recorded using a graduated micrometer in the microscope eyepiece. E m b r y o n i c differentiation was assessed using a point scoring system developed by Brown and Fabro (18). A further assessment of growth was made by estimating the total protein content in each e m b r y o by the method o f L o w r y (19). Results were analysed using Student's t test for all parameters except the number of abnormal embryos, which were compared through X2 test.
RESULTS A summary of the results from culture experiments using serum during various stages o f the menstrual cycle and pregnancy is given in Table I. Rat embryos cultured in 100% rat serum have comparable morphological scores, yolk sac diameters, c r o w n - r u m p lengths, and somite numbers, but significantly reduced protein values (p < 0.01) when compared with 11.5 day in-vivo embryos. Types o f developmental anomalies seen in sera from various groups are shown in Table 2. The majority o f the anomalies consisted of failure o f the cranial end of the neural tube to close and o f the trunk of the embryo to " t u r n " normally (Figure 1). E m b r y o n i c growth and differentiation were almost identical between conceptuses cultured in undiluted rat serum and those cultured in human serum supplemented with glucose and rat serum. Yolk sac diameter and crown-rump length were greater (p < 0.01) in e m b r y o s cultured in 10% rat serum-supplemented human serum compared with
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Reproductive Toxicology
Volume 2, N u m b e r 2, 1988
Table 2. T y p e s o f d e v e l o p m e n t a l anomalies in serum from various groups Open
In vivo 100% rat serum 100% human serum Preovulatory* Postovulatory*
1st trimester* 2nd trimester* 3rd trimester* Postpartum*
neural
Trunk
H e ma -
Anoph-
tubes
abnormalities
tomata
thalmia
-6 ----3 --
-3 --
.
.
.
2 7 -1 2 3 ---
.
1 4 4 2 1 1 -3
1 --1 --
Note: In some cases more than one anomaly appeared in the same embryo. *90% human serum supplemented with 10% rat serum.
those grown in 100% rat serum. The number of conceptuses with developmental anomalies was comparable for those in 10% rat serum-supplemented human serum and those in 100% rat serum. The results from experiments studying the effects of diluting the human serum with varying amounts of rat serum are shown in Table 3. It can be seen that as the percentage of rat serum supplementation is reduced, there is a significant decrease (p < 0.01) in the yolk sac diameter, crown-rump
length, somite number, and protein values, but there are no significant differences in the morphological scores between the three groups where the human serum is supplemented with rat serum. Embryos cultured in 100% human serum supplemented only with glucose have significantly reduced morphological scores (p < 0.02) as well as increased numbers of abnormalities (p < 0.02), chiefly due to abnormal turning and nonclosure of the neural tube.
Table 3. E m b r y o n i c growth in h u m a n serum at various concentration o f rat serum supplementation Growth
parameter
10%
5%
2%
0%
10
8
7
7
110
57
51
50
42.85 0.06
42.21 0.32
40.26 0.68*
No. of
patients No. of embryos
Morphological score S.E.
42.55 0.08
Y o l k sac
diameter (mm) S.E. Crown-rump length (mm) S.E. Somite number S.E. Embryonic protein (p.g) S.E. Abnormal embryos (X2)
4.25 0.03
4.11 0.02**
3.92 0.03**
3.76 0.05
3.42 0.03
3.26 0.04**
3.07 0.03**
2.96 0.05
26.32 0.09
26.71 0.06**
25.33 0.12"*
24.94 0.16"*
212.59 4.99
190.28 3.81"*
163.87 2.70**
2 (0.77)
2 (0.32)
3 (0.01)
132.58 3.51" 10 (5.48*)
*Sig. at p < 0.02 w h e n c o m p a r e d with the values in the preceding column. **Sig. at p < 0.01 w h e n c o m p a r e d with the values in the preceding column.
Rat e m b r y o culture in h u m a n s e r u m • M. ANWAR and F. BECK
139
On gross inspection it was observed that embryos cultured in pure human serum lacked hemoglobin, whereas the conceptuses cultured in supplemented human serum were not anemic. As the concentration of rat serum supplement was decreased, embryonic anemia became more apparent. DISCUSSION
(a)
(b)
Fig. 1. 9.5-day rat embryos cultured for 48 hours. (a) Normal embryo after culture in rat serum, x22. (b) Abnormal embryo after culture in supplemented human serum, x35.
Although rat conceptuses in culture differentiate similarly to those in vivo, as shown by the comparable morphological scores, the protein values are reduced (Table 1). This is probably due to the disturbance occasioned by the explantation procedure, which involves keeping embryos at room temperature for about 30 minutes. It is also possible that the chorio-allantoic placenta may begin to play a significant part in embryonic nutrition sometime during the 12th day of gestation (equivalent to the very end of the culture period), in which case invitro embryos in which this placental system cannot develop would be slightly disadvantaged during the last few hours of culture. Both normal morphogenesis and growth were significantly reduced when human serum was supplemented with glucose only. This suggests that pure human serum, despite the addition of glucose, still lacks some of the factors necessary for the normal growth of rat embryos. However, when human serum is diluted with 10% rat serum, the degree of embryonic growth and morphogenesis obtained is comparable to that obtained in 100% rat serum. Indeed, conceptuses grown in human serum diluted to 90% with 10% rat serum had larger yolk sac diameters and crown-rump lengths than those grown in pure rat serum, possibly because all human sera were supplemented with glucose to 3 mg/mL. The results in Table 3 show that as the rat serum supplementation is reduced, there is a deterioration in the embryo growth-supporting capacity of the human serum. It was observed that although supplementation with 2% rat serum provided sufficient species-specific growth factors for rat embryos to achieve reproducibly normal morphologic differentiation in glucose-supplemented human serum, they do not grow to their full potential. It appears that rat embryos remain growth-retarded when cultured in human serum containing less than 10% rat serum. It was clearly seen that embryos cultured in human serum supplemented with decreasing amounts of rat serum were increasingly lacking in hemoglobin, and this supports the suggestion made by Huxham and Beck (20) that the
140
Reproductive Toxicology
anemia present in rat embryos cultured in human serum is due to a lack of (rat) species-specific serum transferrin. In the experimental work reported here, there were fewer abnormalities (12%) when embryos were grown in glucose-only supplemented human serum than the 41.7% reported by Reti et al. (5). This may be due to the fact that the conceptuses used in our study were standardized in that only late head-fold embryos (approximately 3 somites) were used, whilst Reti et al. (5) explanted all the embryos present from their 9.5 days pregnant rats. Some of these were probably developmentally less advanced than those used here, and our experience indicates that early embryos are more susceptible to certain embryopathic effects than more advanced ones (21). The results presented in Table 1 indicate that when using 90% human serum as the culture medium supplemented with glucose and 10% rat serum, embryonic growth parameters from each experimental group are comparable with those obtained with pure rat serum. Bartlett's (22) test for the homogeneity of variance was performed for protein values in Groups A, B, C, D, E, and F. An F value of 75.78 was obtained. This indicates (p < 0.001) that the groups come from a homogeneous population and that there are no significant differences among the groups. Therefore, under the culture conditions we employed and using the criteria described to assess the embryonic growth and morphogenesis, changing levels of steroid hormones and various other biochemical parameters during the menstrual cycle and advancing pregnancy do not affect embryonic growth. This is a significant observation since it means that, for clinic evaluation, human serum can be obtained at any stage of the menstrual cycle and pregnancy. Furthermore it may also be concluded that in order to achieve embryos with normal morphological scores, human serum should be supplemented with at least 2% rat serum. We suggest that embryo culture experiments in human serum diluted with only 2% rat serum are more likely to reflect the true embryo growth-supporting capacity of human serum than studies that are conducted with higher rat serum supplementation. This is because, if larger amounts of rat serum were added, the species specific growth factors could over-compensate for minor deficiencies within the human serum thus leading to erroneous conclusions in experiments where human serum was only mildly embryopathic. A c k n o w l e d g e m e n t - We are grateful to the Trent Regional Health Authority for a grant in aid of research.
Volume 2, Number 2, 1988
REFERENCES 1. Tanimura T, Shepard TH. Glucose metabolism by rat embryos in-vitro. Proc Exp Biol Med 1970; 135:51-53. 2. New DAT. Development of explanted rat embryos in circulating medium. J Embryol Exp Morph 1967; 17:513--525. 3. Chatot CL, Klein NW, Piatek, J, Pierro LJ. Successful culture of rat embryos in human serum: use in detection of teratogens. Science 1980; 207:1471-1473. 4. Porov VB, Puchkov VE, Ignatieva V. In-vitro development of post-implanted embryos of laboratory rodents in human blood serum. Arkhiv Anatomii Gistologii embryologii 1981; 81(part 11):92-94. 5. Reti LL, Beck F, Bulman S. Culture of 9.5 days rat embryos in human serum supplemented and unsupplemented with rat serum. J Exp Zool 1982; 223:197-199. 6. Lear D, Clark A, et al. Morphological total nucleic acid and total protein analysis of rat embryos cultured in supplemented and unsupplemented human serum. J Anat 1983; 173:285-297. 7. Gupta M, Beck F. Growth of 9.5 days rat embryos in human serum. J Embryol Exp Morph 1983; 76:1-8. 8. Priscott PK. Rat post-implantation embryo culture using heterologous serum. Aust J Exp Med Sci 1983; 61:Part l, 47-55. 9. Steele CE. Human serum as a culture medium for rat embryos. Experientia 1985: 41:1601-1603. 10. Chatot CL, Klein NW. Teratogenic activity of serum from human epileptic subjects. Studies by rat embryo cultures. Teratology 1981; 23:30A. 11. AI-Alousi LA. Rat Embryonic Nutrition. University of Leicester; 1983. PhD. Thesis. 12. Cockroft DL. Nutrient requirements of rat embryos undergoing organogenesis in-vitro. J Reprod Fertil 1979; 57:505510. 13. Beck F, Huxham MI, Gulamhusein AP. Growth of rat embryos in the serum of alcohol drinkers: Fetal Alcohol Syndrome. 'Mechanism of Alcohol Damage in in Utero,' CIBA Foundation Symposium 105, 1984:218-233. New York: John Wiley. 14. Tam PLT et al. A correlative study on the embryotrophic property of patients' serum and the outcome of in vitro fertilation of human oocytes. Fertillity and Sterility 1987; 5:834839. 15. Hytten FE, Leitch 1. The physiology of human pregnancy. 2nd ed. Oxford: Blackwell Scientific Publications; 1971. 16. Buckley SKL, Steele CE, New DAT. In vitro development of early post-implantation rat embryos. Dev Biol, 1978; 65:396-403. 17. New DAT. Whole embryo culture and the study of mammalian embryos during organogenesis. Biol Rev 1978; 53:81122. 18. Brown NA, Fabro S. Quantitation of rat embryonic development in vitro. A morphological scoring system. Teratology 1981; 24:65-78. 19. Lowry DH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the folin phenol reagent. J Biol Chem 1951; 193:265-275. 20. Huxham MI, Beck F. Maternal transferrin uptake by the transfer across the visceral yolk sac of the early postimplantation rat conceptus in vitro. Dev Biol 1985; 110:7583. 21. Clode AM, Pratten MK, Beck F. Stage dependent effect of ethanol in 9.5 day rat embryos grown in ethanol and the role played by the concomitant rise in osmolality. Teratology 1987; 35:395-403. 22, Bartlett MS. Properties of sufficiency and statistical tests. Proe R Soc A 1937; 160:268-282.
0890-6238/88$3.00 + .00 Copyright (~ 1988PergamonPress pie
Printed in the U.S.A.
EVALUATION
OF HUMAN SERUM OF RAT EMBRYONIC
AS A CULTURE DEVELOPMENT
MEDIUM FOR STUDIES IN VITRO
MOHAMMAD ANWAR* and FELIX BECKt Departments of ?Anatomy and *Obstetrics & Gynaecology, University of Leicester, University Road, Leicester, England Abstract m The normal growth of rat embryos cultured in human serum supplemented with glucose and 10% rat serum is of potential use in the assessment of teratogenic risk in man. Before this technique can be widely applied, it is necessary to know whether the stages of the menstrual cycle at which a serum sample is tested materially affect the results obtained. Moreover, in order to achieve reproducible conditions we have found it necessary to add a minimal amount of rat serum to the human serum used for culture, but unnecessarily high levels of rat serum supplementation could over-compensate for any growth factor deficiency in human serum. Here we report that culture of rat conceptuses gives similar results irrespective of whether human first, second, or third trimester pregnancy serum, postnatal serum, or serum at various stages of the menstrual cycle is used. We also report that addition of 2% rat serum supplement is sufficient to achieve reproducible rat embryonic growth and differentiation in human serum. Key Words: Teratology, Whole e m b r y o culture, In vitro drug evaluation.
INTRODUCTION
embryopathic properties of serum obtained from epileptic patients on various anticonvulsants. A review of the original data reported by Chatot et al. (3) shows that 20% of the embryos cultured in normal human serum developed abnormally. This makes it difficult to use the original method in critical experiments because no acceptable " n o r m a l " baseline for controls is available. In an attempt to achieve reproducible rat embryonic growth and differentiation, Reti et al. (5) diluted human serum with 10% rat serum and supplemented glucose to a final concentration o f 3 mg/mL. They reported that embryonic growth and differentation as measured by protein levels and morphological assessment was comparable with that occurring in pure rat serum and also that the embryos had significantly fewer developmental anomalies when compared with those grown in glucose-only supplemented human serum. L e a r et al. (6) and Gupta and Beck (7), using the same conditions, also reported a significant improvement in morphological scores, hemoglobin contents, and protein values, with a lower range o f abnormalities when rat conceptuses were cultured in human serum that had been supplemented with glucose and 10% rat serum. These authors proposed that a small aliquot of rat serum added to the human serum provides only the species-specific growth factors required by the rat embryos to differentiate normally. In support of
In 1970 Tanimura and Shepard (1) reported that N e w ' s (2) roller bottle in-vitro embryo culture technique could be used to grow postimplantation rat conceptuses in human serum. Since then many workers, including Chatot et al. (3), Porov et al. (4), Reti et al. (5), L e a r et al. (6), Gupta et al. (7), Priscott (8), and Steele (9), have performed in-vitro e m b r y o culture experiments in undiluted human serum and have published inconsistent results. The inconsistencies within the reported results include unacceptably high levels of developmental anomalies as well as a varying protein content among cultured embryos. C h a t o t et al. (3) suggested that postimplantation rat conceptuses developed more successfully when undiluted human serum was supplemented with glucose to a final concentration of 3 mg/mL. Glucose supplementation was thought to be necessary, as the glucose level in rat serum (1.8 mg/mL) is higher than the human serum level (0.8 mg/mL). These authors proposed that rat whole e m b r y o culture could be used in a clinical context to screen for embryopathic factors in human serum. A year later Chatot et al. (10) applied this method to test the A d d r e s s c o r r e s p o n d e n c e to: Dr. M. A n w a r , D e p a r t m e n t of Obstetrics & Gynaecology, Leicester Royal Infirmary, Leicester, England. Received 16 February 1988; Accepted 4 July 1988. 135
136
Reproductive Toxicology
this hypothesis, A1-Alousi (11) has shown that 10% rat serum diluted with 90% Cockroft's (12) balanced salt solution and appropriately supplemented with glucose and vitamins was insufficient to support embryonic growth. From this it may be inferred that the growth factors in the rat serum (lacking in human serum) are required for the embryos to grow and differentiate to their full capacity in human serum. Beck et al. (13) used this modified technique to demonstrate the teratogenic properties of human serum obtained from volunteers who had consumed alcohol under experimental conditions. It is clear that if the in-vitro embryo culture technique is to be applied in a clinical context the conditions under which the experiments are performed must be standardized. The maternal "milieu interieur" during the menstrual cycle and pregnancy is altered to create conditions favorable for implantation and development. Apart from a changing hormonal profile, there are widespread changes in the constituents of serum during pregnancy, including a fall in serum proteins, pH, urea, and electrolytes, consequently leading to reduced osmolality as discussed by Hytten and Leitch (15). These changes could alter the embryo growthsupporting capacity of human serum. It is thus important to define the stage of the menstrual cycle and of gestation at which the serum is potentially suitable for in-vitro culture. Although Buckley et al. (16) has reported that equally satisfactory results were obtained when serum from male, nonpregnant, and pregnant rats was used as a culture medium, no comparable studies have been performed using serum obtained from humans. Furthermore, if human serum is supplemented with rat serum, there is no consensus regarding the optimum amount of rat serum required for standard embryonic growth and differentiation. If biologically and clinically meaningful conclusions are to be drawn from the culture of rat embryos in human serum, it is important that supplementation with rat serum is kept to a minimum. The addition of unnecessarily high levels of rat serum makes it possible that deficiencies present in the human serum could be compensated by the added rat serum. MATERIALS AND METHODS
Selection of patients and the preparation of serum Blood was obtained from female medical students, nursing staff, patients attending antenatal clinics, and mothers in the postnatal wards between the hours of 0900 and 1000, after their normal breakfasts. Apart from patients on hematinic supple-
Volume 2, Number 2, 1988
mentation, those volunteers on oral contraceptives or any other drug therapy were excluded. Blood samples were obtained from women who, according to their last normal menstrual period, were in: 1) The first half of the menstrual cycle (day 5 to 14) 2) The second half of the menstrual cycle (day 15 to 28) 3) The first trimester of pregnancy (up to 14th gestational week) 4) The second trimester of pregnancy (15th to 28th gestational week) 5) The third trimester of pregnancy (29th to 42nd gestational week) 6) The post-delivery period (up to 5 days) For experiments to study the effects of supplementing human serum with various concentrations of rat serum, blood samples were obtained only from women in the second half of their menstrual cycle. Thirty mL human blood samples were centrifuged immediately after venesection and treated according to the method of New (17). Prior to use, human serum was always supplemented with glucose to a final concentration of 3 mg/mL and with an appropriate amount of rat serum; 0.02 mL of antibiotics (stock solution containing streptomycin 5000/zg/mL and penicillin 5000 Iu/mL) were added per mL of all culture media. Cultures were gassed initially with a mixture of 5% 02, 5% CO2, and 90% N2. After 24 hours this was replaced with 20% 02, 5% CO2 and 75% Nz and after 42 hours with 40% 02, 5% CO2 and 55% N2. For control experiments, conceptuses were cultured in pooled rat serum obtained from several pregnant animals.
Explantation of rat conceptuses Conceptuses were obtained from Wistar rats at a later time of 1100 hours of the 10th day of gestation (timed from midnight preceding the morning on which vaginal plugs were observed). They were removed under aseptic conditions in Hank's balanced salt solution and, in order to standardize the experiment, only those at the late head-fold stage of development (3 somites) were selected for the culture experiments. Embryo culture Conceptuses from each litter were randomly divided into experimental and control groups. Embryos were cultured at 37 °C for 48 hours in rotating
137
Rat embryo culture in human serum • M. ANWARand F. BECK Table 1. Embryonic growth in pure and supplemented (10% rat serum) human serum 10% rat serum supplementation Growth parameter
invivo
100% rat serum
100% Postovul hum serum
(A) Preovul
(B) Postovul
7
10
10
50
110
110
(D) 2ndtrim
(E) 3rdtrim
(F) Postnatal
9
10
9
9
90
95
90
90
42.56 0.13
42.66 0.14
42.75 0.11
42.45 0.19
(C) lsttrim
No. of patients No. of embryos
25
Morphological score S.E.
42.96 0,03
42.64 0.08
40.26 0.68**
3,94 0.06
3.90 0.04
3.76 0.05**
4.14 0.03**
4.25 0.03**
4.16 0.04
4.28 0.02**
4.25 0.04**
4.00 0.04**
3.33 0.05
3.33 0.04
2.96 0.05**
3.42 0.02**
3.42 0.03*
3.43 0.04
3.49 0.02**
3.43 0.03
3.26 0.03
26.86 0,09
26.50 0.09
24.94 0.16"*
289.91 13.50
215.02 4.15"*
132.58 3.51"*
Yolk sac diameter (mm) S.E. Crown-rump length (ram) S.E. Somite number S.E. Protein (/xg) S.E. Abnormal embryos
(Xz)
0
106
3
l0 (5.48**)
42.55 0.07
42.55 0.08
26.32 0.09
26.37 0.13
26.36 0.14
26.48 0.10
25.94 0.10
209.29 4.08
212.59 4.99
203.78 5.67
205.59 4.13
201.99 5.33
203.40 4.67
4 (0.1)
3 (0.01)
3 (0.04)
3 (0.02)
4 (0.35)
26.12 0.12"
4 (0.35)
ovul = ovulatory; trim -- trimester; No. = number of embryos. *p < 0.02 when compared with embryonic growth in whole rat serum. **p < 0.01 when compared with embryonic growth in whole rat serum.
glass bottles according to the method o f N e w (17). E a c h bottle contained 5 conceptuses in 5 m L of the culture medium. On average, 10 conceptuses per human serum sample were cultured. H u m a n serum was supplemented with glucose and various concentrations of rat serum. Control experiments were performed in 100% rat serum.
Assessment of embryonic growth and development At the end o f the culture period conceptuses were examined under a dissecting microscope. As a measure o f development their maximum yolk sac diameters and crown/rump lengths were recorded using a graduated micrometer in the microscope eyepiece. E m b r y o n i c differentiation was assessed using a point scoring system developed by Brown and Fabro (18). A further assessment of growth was made by estimating the total protein content in each e m b r y o by the method o f L o w r y (19). Results were analysed using Student's t test for all parameters except the number of abnormal embryos, which were compared through X2 test.
RESULTS A summary of the results from culture experiments using serum during various stages o f the menstrual cycle and pregnancy is given in Table I. Rat embryos cultured in 100% rat serum have comparable morphological scores, yolk sac diameters, c r o w n - r u m p lengths, and somite numbers, but significantly reduced protein values (p < 0.01) when compared with 11.5 day in-vivo embryos. Types o f developmental anomalies seen in sera from various groups are shown in Table 2. The majority o f the anomalies consisted of failure o f the cranial end of the neural tube to close and o f the trunk of the embryo to " t u r n " normally (Figure 1). E m b r y o n i c growth and differentiation were almost identical between conceptuses cultured in undiluted rat serum and those cultured in human serum supplemented with glucose and rat serum. Yolk sac diameter and crown-rump length were greater (p < 0.01) in e m b r y o s cultured in 10% rat serum-supplemented human serum compared with
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Volume 2, N u m b e r 2, 1988
Table 2. T y p e s o f d e v e l o p m e n t a l anomalies in serum from various groups Open
In vivo 100% rat serum 100% human serum Preovulatory* Postovulatory*
1st trimester* 2nd trimester* 3rd trimester* Postpartum*
neural
Trunk
H e ma -
Anoph-
tubes
abnormalities
tomata
thalmia
-6 ----3 --
-3 --
.
.
.
2 7 -1 2 3 ---
.
1 4 4 2 1 1 -3
1 --1 --
Note: In some cases more than one anomaly appeared in the same embryo. *90% human serum supplemented with 10% rat serum.
those grown in 100% rat serum. The number of conceptuses with developmental anomalies was comparable for those in 10% rat serum-supplemented human serum and those in 100% rat serum. The results from experiments studying the effects of diluting the human serum with varying amounts of rat serum are shown in Table 3. It can be seen that as the percentage of rat serum supplementation is reduced, there is a significant decrease (p < 0.01) in the yolk sac diameter, crown-rump
length, somite number, and protein values, but there are no significant differences in the morphological scores between the three groups where the human serum is supplemented with rat serum. Embryos cultured in 100% human serum supplemented only with glucose have significantly reduced morphological scores (p < 0.02) as well as increased numbers of abnormalities (p < 0.02), chiefly due to abnormal turning and nonclosure of the neural tube.
Table 3. E m b r y o n i c growth in h u m a n serum at various concentration o f rat serum supplementation Growth
parameter
10%
5%
2%
0%
10
8
7
7
110
57
51
50
42.85 0.06
42.21 0.32
40.26 0.68*
No. of
patients No. of embryos
Morphological score S.E.
42.55 0.08
Y o l k sac
diameter (mm) S.E. Crown-rump length (mm) S.E. Somite number S.E. Embryonic protein (p.g) S.E. Abnormal embryos (X2)
4.25 0.03
4.11 0.02**
3.92 0.03**
3.76 0.05
3.42 0.03
3.26 0.04**
3.07 0.03**
2.96 0.05
26.32 0.09
26.71 0.06**
25.33 0.12"*
24.94 0.16"*
212.59 4.99
190.28 3.81"*
163.87 2.70**
2 (0.77)
2 (0.32)
3 (0.01)
132.58 3.51" 10 (5.48*)
*Sig. at p < 0.02 w h e n c o m p a r e d with the values in the preceding column. **Sig. at p < 0.01 w h e n c o m p a r e d with the values in the preceding column.
Rat e m b r y o culture in h u m a n s e r u m • M. ANWAR and F. BECK
139
On gross inspection it was observed that embryos cultured in pure human serum lacked hemoglobin, whereas the conceptuses cultured in supplemented human serum were not anemic. As the concentration of rat serum supplement was decreased, embryonic anemia became more apparent. DISCUSSION
(a)
(b)
Fig. 1. 9.5-day rat embryos cultured for 48 hours. (a) Normal embryo after culture in rat serum, x22. (b) Abnormal embryo after culture in supplemented human serum, x35.
Although rat conceptuses in culture differentiate similarly to those in vivo, as shown by the comparable morphological scores, the protein values are reduced (Table 1). This is probably due to the disturbance occasioned by the explantation procedure, which involves keeping embryos at room temperature for about 30 minutes. It is also possible that the chorio-allantoic placenta may begin to play a significant part in embryonic nutrition sometime during the 12th day of gestation (equivalent to the very end of the culture period), in which case invitro embryos in which this placental system cannot develop would be slightly disadvantaged during the last few hours of culture. Both normal morphogenesis and growth were significantly reduced when human serum was supplemented with glucose only. This suggests that pure human serum, despite the addition of glucose, still lacks some of the factors necessary for the normal growth of rat embryos. However, when human serum is diluted with 10% rat serum, the degree of embryonic growth and morphogenesis obtained is comparable to that obtained in 100% rat serum. Indeed, conceptuses grown in human serum diluted to 90% with 10% rat serum had larger yolk sac diameters and crown-rump lengths than those grown in pure rat serum, possibly because all human sera were supplemented with glucose to 3 mg/mL. The results in Table 3 show that as the rat serum supplementation is reduced, there is a deterioration in the embryo growth-supporting capacity of the human serum. It was observed that although supplementation with 2% rat serum provided sufficient species-specific growth factors for rat embryos to achieve reproducibly normal morphologic differentiation in glucose-supplemented human serum, they do not grow to their full potential. It appears that rat embryos remain growth-retarded when cultured in human serum containing less than 10% rat serum. It was clearly seen that embryos cultured in human serum supplemented with decreasing amounts of rat serum were increasingly lacking in hemoglobin, and this supports the suggestion made by Huxham and Beck (20) that the
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anemia present in rat embryos cultured in human serum is due to a lack of (rat) species-specific serum transferrin. In the experimental work reported here, there were fewer abnormalities (12%) when embryos were grown in glucose-only supplemented human serum than the 41.7% reported by Reti et al. (5). This may be due to the fact that the conceptuses used in our study were standardized in that only late head-fold embryos (approximately 3 somites) were used, whilst Reti et al. (5) explanted all the embryos present from their 9.5 days pregnant rats. Some of these were probably developmentally less advanced than those used here, and our experience indicates that early embryos are more susceptible to certain embryopathic effects than more advanced ones (21). The results presented in Table 1 indicate that when using 90% human serum as the culture medium supplemented with glucose and 10% rat serum, embryonic growth parameters from each experimental group are comparable with those obtained with pure rat serum. Bartlett's (22) test for the homogeneity of variance was performed for protein values in Groups A, B, C, D, E, and F. An F value of 75.78 was obtained. This indicates (p < 0.001) that the groups come from a homogeneous population and that there are no significant differences among the groups. Therefore, under the culture conditions we employed and using the criteria described to assess the embryonic growth and morphogenesis, changing levels of steroid hormones and various other biochemical parameters during the menstrual cycle and advancing pregnancy do not affect embryonic growth. This is a significant observation since it means that, for clinic evaluation, human serum can be obtained at any stage of the menstrual cycle and pregnancy. Furthermore it may also be concluded that in order to achieve embryos with normal morphological scores, human serum should be supplemented with at least 2% rat serum. We suggest that embryo culture experiments in human serum diluted with only 2% rat serum are more likely to reflect the true embryo growth-supporting capacity of human serum than studies that are conducted with higher rat serum supplementation. This is because, if larger amounts of rat serum were added, the species specific growth factors could over-compensate for minor deficiencies within the human serum thus leading to erroneous conclusions in experiments where human serum was only mildly embryopathic. A c k n o w l e d g e m e n t - We are grateful to the Trent Regional Health Authority for a grant in aid of research.
Volume 2, Number 2, 1988
REFERENCES 1. Tanimura T, Shepard TH. Glucose metabolism by rat embryos in-vitro. Proc Exp Biol Med 1970; 135:51-53. 2. New DAT. Development of explanted rat embryos in circulating medium. J Embryol Exp Morph 1967; 17:513--525. 3. Chatot CL, Klein NW, Piatek, J, Pierro LJ. Successful culture of rat embryos in human serum: use in detection of teratogens. Science 1980; 207:1471-1473. 4. Porov VB, Puchkov VE, Ignatieva V. In-vitro development of post-implanted embryos of laboratory rodents in human blood serum. Arkhiv Anatomii Gistologii embryologii 1981; 81(part 11):92-94. 5. Reti LL, Beck F, Bulman S. Culture of 9.5 days rat embryos in human serum supplemented and unsupplemented with rat serum. J Exp Zool 1982; 223:197-199. 6. Lear D, Clark A, et al. Morphological total nucleic acid and total protein analysis of rat embryos cultured in supplemented and unsupplemented human serum. J Anat 1983; 173:285-297. 7. Gupta M, Beck F. Growth of 9.5 days rat embryos in human serum. J Embryol Exp Morph 1983; 76:1-8. 8. Priscott PK. Rat post-implantation embryo culture using heterologous serum. Aust J Exp Med Sci 1983; 61:Part l, 47-55. 9. Steele CE. Human serum as a culture medium for rat embryos. Experientia 1985: 41:1601-1603. 10. Chatot CL, Klein NW. Teratogenic activity of serum from human epileptic subjects. Studies by rat embryo cultures. Teratology 1981; 23:30A. 11. AI-Alousi LA. Rat Embryonic Nutrition. University of Leicester; 1983. PhD. Thesis. 12. Cockroft DL. Nutrient requirements of rat embryos undergoing organogenesis in-vitro. J Reprod Fertil 1979; 57:505510. 13. Beck F, Huxham MI, Gulamhusein AP. Growth of rat embryos in the serum of alcohol drinkers: Fetal Alcohol Syndrome. 'Mechanism of Alcohol Damage in in Utero,' CIBA Foundation Symposium 105, 1984:218-233. New York: John Wiley. 14. Tam PLT et al. A correlative study on the embryotrophic property of patients' serum and the outcome of in vitro fertilation of human oocytes. Fertillity and Sterility 1987; 5:834839. 15. Hytten FE, Leitch 1. The physiology of human pregnancy. 2nd ed. Oxford: Blackwell Scientific Publications; 1971. 16. Buckley SKL, Steele CE, New DAT. In vitro development of early post-implantation rat embryos. Dev Biol, 1978; 65:396-403. 17. New DAT. Whole embryo culture and the study of mammalian embryos during organogenesis. Biol Rev 1978; 53:81122. 18. Brown NA, Fabro S. Quantitation of rat embryonic development in vitro. A morphological scoring system. Teratology 1981; 24:65-78. 19. Lowry DH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the folin phenol reagent. J Biol Chem 1951; 193:265-275. 20. Huxham MI, Beck F. Maternal transferrin uptake by the transfer across the visceral yolk sac of the early postimplantation rat conceptus in vitro. Dev Biol 1985; 110:7583. 21. Clode AM, Pratten MK, Beck F. Stage dependent effect of ethanol in 9.5 day rat embryos grown in ethanol and the role played by the concomitant rise in osmolality. Teratology 1987; 35:395-403. 22, Bartlett MS. Properties of sufficiency and statistical tests. Proe R Soc A 1937; 160:268-282.