*Baystate Medical Center, Tufts University School of Medicine, Department of Pathology, 759 Chestnut Street, Springfield, MA 01119

Abbreviated Title: Primary in-situ culture vs. Secondary trypsinized culture

Indexing terms: Spontaneous miscarriages, Culture methods, Trypsinized culture, In-Situ culture, Fetal karyotype

Correspondence to: Rizwan Naeem, M.D., FACMG., Baystate Medical Center, Tufts University School of Medicine, 759 Chestnut Street, Springfield, MA 01199, Phone: 413 794-4363, Fax: 413 794-5481, Email: rizwan.naeem@bhs.org

Affiliation and Address for both authors and address for reprints: Laboratory Genetics, Department of Pathology, Baystate Medical Center, 759 Chestnut Street, Springfield, MA 01199

Acknowledgements: We would like to acknowledge Dr. Solveig Pfleuger and the cytogenetic laboratory staff, at Baystate Medical Center for their contributions.

Abstract

Objectives:
Primary in-situ culture (PIC) and secondary trypsinized culture (STC) are the two currently used methods for culturing chorionic villi in order to cytogenetically evaluate products of conception (POC) from spontaneous miscarriages. We compare these culture techniques in our laboratory over a period of seven years to evaluate fetal karyotype yield and maternal cell contamination.

Methods:
Data from a total of 2077 cases from 1992-1999 was entered into a data entry program created in Epi Info version 6. Analysis, using the chi square test of significance, was performed in the same program.

Results:
Our data demonstrated a statistically significant excess of normal female karyotype detected by the STC method and a statistically significant excess of abnormal karyotypes detected by the PIC method. We attribute these findings to the greater risk of maternal cell contamination with the STC method.

Conclusions:
We conclude that the PIC method is more accurate in detecting the fetal karyotype and the STC method has a higher risk of maternal cell contamination. We suggest that the PIC method should be adopted as the method of choice when evaluating POC by culturing chorionic villi.

Introduction

It is estimated that about sixty to seventy percent of human conceptions are lost through the course of gestation (Dejmek, 1992). The majority of these losses occur prior to implantation or within the first four weeks of pregnancy (Wilcox, 1988). An estimated fifteen to twenty five percent of spontaneous miscarriages (SM) occur between the 6^th and 28^th weeks of gestation as clinically recognized abortions (French, 1967) making pregnancy loss a considerable clinical and economical issue.

Spontaneous pregnancy loss can be divided into either maternal or fetal causes (Hacker and Moore, 1998). The most important fetal factor is a significant genetic abnormality of the conceptus (Kline, 1985). Cytogenetic investigations of products of conception show that approximately 40-50% of early pregnancy loss results from unbalanced chromosomal abnormalities in the conceptus (Boue, 1985; Jacobs, 1987; Ohno, 1991). Therefore, these cytogenetic investigations provide information about the cause of miscarriage and may aid in the prenatal diagnosis of subsequent pregnancies (Ohno, 1991). The cytogenetic characterization of POC when normal helps in the identification of non-karyotypic genetic causes of repeated pregnancy loss. These may include parental HLA sharing (Ober, 1995), mutated early developmental genes (Rossant, 1989) and situations of non-complementation by uniparental disomy (Shaffer, 1998). From a scientific perspective cytogenetic investigations provide information on both the frequency and type of chromosomal abnormalities in different populations, as well as their etiology and recurrence risk (Eiben, 1990).

Advances in the technology have resulted in an evolution of culture techniques used for processing POC for karyotyping. Initially, culture of POC involved taking samples from cord blood, amniotic/chorionic membrane or fetal skin. Samples were then subjected to conventional tissue culture. These culture techniques were laborious and carried a risk for contamination, culture failure and sometimes selective growth of maternal cells (Eiben, 1990). Progression was then made to culture of chorionic villi (Cheung S, 1987) and later to direct chromosome preparations from chorionic villi (Ohno, 1991; Eiben, 1990; Songster, 1992; Gardo, 1992; Eiben, 1987).

Both the culture of chorionic villi and direct preparation of metaphases from chorionic villi have their advantages and disadvantages. The former yields better quality metaphases and is less sensitive to processing time, whereas the latter is less laborious, yields faster results and is unlikely to be subject to maternal cell contamination (Cheung, 1987; Griffin, 1997).

Our lab has been using the method of culturing chorionic villi for the purpose of karyotyping POC. Over the last several years, we have been using two distinct protocols or methods for culturing chorionic villi from POC: the PIC and the STC methods. In the PIC method, the villi are desegregated using enzymatic digestion with trypsin and collagenase for single cell suspension yield. This single cell suspension is then set up on coverslips for primary cultures, which yield cells used for metaphase preparations. In the STC method, pieces of villi are first set up for long-term culture in a culture flask and subsequently trypsinized as secondary cultures for metaphase preparations.

The objective of this study was to determine which of the above two culture methods is less likely to give rise to maternal cell contamination, this being a concern with chorionic villus cultures (Cheung, 1987; Griffin, 1997), and hence is more accurate in detecting fetal karyotypes.

Materials and Methods

Culture methods
The data for this study was obtained from patients’ cytogenetic charts, on samples that were received in our cytogenetics laboratory from 1992 through 1999. All charts were reviewed retrospectively for culture technique used, other clinical and demographic information and final karyotype.

In both methods, POC from spontaneous miscarriages were routinely collected in clean, dry, airtight containers preferably in sterile isotonic saline. Samples were then sent to the cytogenetics laboratory, where a trained technologist dissected and selected placental chorionic villi from the gestational sac under sterile conditions. Possible maternal tissue was identified and removed. Only samples with visual presence of villi were included in the study.

In the PIC method, selected villi were first incubated in a mixture of 8 ml of Hank’s balanced salt solution and 2 ml of Pen-Strep (Gibco BRL Cat. No 10378) for 5 minutes. Villi were then cut into 4 or 5 small pieces and treated with 2 ml of trypsin EDTA (Gibco BRL Cat. No 25300-054) for one hour at 37 degrees Celsius. Following incubation, trypsin was removed carefully and 2 ml of collagenase CLS type III 100U/ml (Worthington Biochemical Cat. No 4128) was added for 30 minutes at 37 degrees Celsius. Subsequently tissue culture media was added and mixed with a pipette to make an even suspension of 3-4 ml. Six to eight cover slips were then prepared with this mixture of cell suspension and tissue culture media resulting in a total volume of 0.5 ml per cover slip. These cover slips were placed in a humidified carbon dioxide incubator for culture. After 48 hours, 2 ml of fresh tissue culture media (Alpha MEM Gibco Cat. No 12571-063) with 10% fetal calf serum was added to the edge of each cover slip. The cultures received fresh tissue culture media on day 4 and then on every other day thereafter, until ready for chromosome harvest.

In the STC method, after incubation in 8 ml of Hank’s balanced salt solution and 2 ml of Pen-Strep for 5 minutes, villi were minced and evenly spread within the T25 flask as an explant culture. After overnight incubation, fresh media was added. These cultures then received fresh tissue culture media, on day 4 and then on every other day thereafter. When ready, this T25 explant culture flask was trypsinized for secondary cultures on the cover slips. These secondary cultures from the cover slips were subsequently harvested for cytogenetic analysis.

In both methods, 50 micro liters of a 50:50 mixture of colcemid and BRDU was added overnight for chromosome harvest. The working solution was prepared by mixing 0.1gm of BRDU (Sigma Cat. No B-5002), 33 ml 0.8% Sodium Citrate, 30 ml of Hank’s balanced salt solution and 3.5 ml of Colcemid (Gibco Cat. No 15210-016).

Data Analysis
Records of 2077 consecutive samples of products of conception referred to The Baystate Medical Center, Cytogenetic Laboratory between 1992 and 1999 were analyzed. Data from these records was initially recorded on a specially designed data entry form, which was then entered into a data entry program created in Epi Info, Version 6 (Center of Disease Control website). Statistical analysis was performed in the same software program using the Chi Square test of significance. The alpha value was chosen to be 0.05. Complete hydatidiform moles and cases where no villi were found by visual evaluation were excluded from this study, as many of them would have been resulted in normal female karyotypes (Szulman, 1978; Hassold, 1980).

Results

From a total of 2077 cases, 1587 cases were selected for final analysis, as the rest of the case records did not have complete information on culture process.

Out of the 1587 samples analyzed, 948 (59.7%) were processed by the PIC method and 639 (40.3%) were processed by the STC method.

The results showed 896 (56.5%) normal karyotypes and 691 (43.5%) abnormal karyotypes. Amongst the 896 normal results, 514 (57.4%) were female and 382 (42.6%) were male karyotypes.

Table 1 illustrates the number of normal male and female karyotypes detected with the PIC and STC method respectively. There was a statisitically significant (Chi square = 8.83, p<0.05) excess of normal female karyotypes detected with the STC method as compared to the PIC method.

Amongst the abnormal karyotypes, there were 115 (16.6%) cases of triploidies, 28 (4.1%) cases of tetraploidies, 381 (55.1%) cases of single trisomies, 22 (3.2%) cases of double trisomies, 5 cases of (0.7%) multiple trisomies, 88 (12.7%) cases of monosomies and 52 other abnormalities including deletions, reciprocal and robertsonian translocations, isochromosomes, derivative chromosomes and mosaicisms.

The percentage of all abnormalities detected with the PIC method was significantly higher (Chi square= 81.09, p<0.05) than the percentage of all abnormalities detected with the STC method.

There were 9 balanced translocations amongst the 691 abnormal karyotypes. These cytogenetically balanced translocations were excluded from further analysis because either one of the parents was a carrier or the parental karyotypes were not available.

Amongst the monosomies, there were 83 cases (94.3%) of monosmy X, 2 cases (2.3%) of monosomy 14, 2 cases (2.3%) of monosomy 21 and one case (1.1%) of monosomy 1.

The mean maternal age in the study population was 35.48 years +/- 6.8 years accompanied by a mean gestational age of 14.9 weeks +/- 7.6 weeks.

The number of common single trisomies detected with each culture method is shown in table 3. Chromosomes 16, 21, 22 and 5 were the most commonly observed chromosomes in single trisomies. All chromosomes were observed in single trisomies with the exception of chromosome 11.

Details of double and multiple trisomies are listed in table 2.

Discussion

Our results demonstrate, that the PIC method is more accurate for the cytogenetic evaluation of fetal karyotype from placental villus cultures than the STC method. The STC method has a higher risk for maternal cell contamination and hence a lower chance of detecting true fetal karyotype. Furthermore, the STC method results in a longer turn around time for cytogenetic results, due to initiation and processing of secondary cultures, in contrast to the PIC method.

Currently most cytogenetic laboratories use either one or both methods depending upon the time and number of trained personnel available. These two methods of culturing chorionic villi have not been previously compared in terms of their accuracy for detecting true fetal karyotypes and the risk of maternal cell contamination due to the culture process.

We have used sex ratio as an index of maternal cell contamination in accordance with literature (Eiben B, 1990; Griffin DK, 1997). We maintain that our patient population was homogenous (as determined by evaluation of demographic data from charts) and theoretically we should have obtained statistically similar sex ratios for samples cultured by both the STC and the PIC method. However there was a statistically significant difference between the two sex ratios obtained with the STC and the PIC method respectively (Chi square= 8.83, p value<0.05). The most likely explanation, as is discussed below is alteration of the sex ratio obtained by the STC method either due to contamination and or selective growth of maternal cells.

In our study, a statistically significant excess of normal female karyotypes was detected with the STC method in comparison to the PIC method. We attribute this observation to the contamination and/or selective growth of trypsinized cultures (STC method) with maternal cells due to the growth advantage available to maternal cells in this long-term culture process.

Another observation is a statistically significant excess of karyotypic abnormalities found with the PIC method as compared to the STC method. We attribute this to the fact that in the PIC method, the use of collagenase promotes cell disassociation, which disperses villous core. This dispersion promotes rapid growth of fetal cells and reduces the level of maternal cell contamination to less than 0.5% (Songster, 1992). A harvest from primary culture then yields a high percentage of fetal cells.

It is known that the risk of maternal cell contamination is much higher with the tissue culture method, than with the method of direct preparation of metaphases from chorionic villi (Bartels, 1990). After reviewing literature, we compared the sex ratio for samples processed by the STC method (prone to maternal cell contamination) with the sex ratio obtained from data, pooled from studies using the tissue culture method to process chorionic villi, as shown in table 4. We also compared the sex ratio for samples processed by the PIC method (less prone to maternal cell contamination) with the sex ratio obtained from data, pooled from studies relying on direct preparation of metaphases from chorionic villi as illustrated in table 5.

There was a statistically significant difference (Chi square = 17.12, p value> 0.05) between the sex ratio obtained from our study, using the STC method, and that obtained from the pooled data, shown in table 4. The lower sex ratio in our study can be explained either by a higher rate of maternal cell contamination or by inter-study variation (some of which could be attributed to the different types of tissues used for culture in the studies quoted). The latter hypothesis is lent some support by the fact, that the first three studies listed in table 4 used fetal tissues such as skin and lymphocytes for karyotyping, whereas the last two studies quoted in the table cultured chorionic villi.

There was no statistically significant difference (Chi square = 3.78, p value < 0.05) between the sex ratio obtained using the PIC method and that obtained from data pooled from studies relying on direct preparation of metaphases from chorionic villi (table 5). Using sex ratio as an index of maternal cell contamination (Griffin, 1997), it can be argued that the PIC method of culturing chorionic villi poses as little risk for maternal cell contamination as the direct preparation of metaphases from chorionic villi.

In conclusion, the STC method of culturing chorionic villi decreases the chances of detecting a true fetal karyotype because it increases the risk of contamination with maternal cells in the tissue culture process when compared to the PIC method. The PIC method appears to be the better choice for culturing placental villi from spontaneous miscarriages and should be adopted as the routine protocol for culturing. It arguably combines the benefits of better metaphase preparation to be had from culturing chorionic villi, while reducing maternal cell contamination to a minimum (Griffin, 1997). We suggest that if STC method is used, the results of a normal female karyotype be interpreted with caution, due to the increased chance of maternal cell contamination.

The findings of this study need to be corroborated by an experimental study in which each POC sample is cultured by both the PIC and STC methods and the results compared. This will do away with the limitations of a retrospective study design and help arrive at definitive conclusions. This study also raises an interesting study question for cytogenetic laboratories: "Do we need to trypsinize secondary culture when there are less than 15 cells available from primary culture?"

For personal use. Only reproduce with permission from SIEP.

References

Bartels I, Hansmann I, Eiben B (1990): Excess of Females in Chromosomally Normal Spontaneous Abortuses. Am J Med Genet 35:297-298.

Boue A, Boue J, Gropp A (1985): Cytogenetics of pregnancy wastage. In Harris H, Hirschhorn K (eds) Advances in Human Genetics. Vol 14. Plenum, New York: 1-57.

Cheung SW, Crane JP, Beaver HA, Burgess BS (1987): Chromosome Mosaicism and Maternal Cell Contamination in Chorionic Villi. Prenat Diagn 7:535-542.

Creasy MR, Crolla JA, Alberman ED (1976): A cytogenetic study of human spontaneous abortions using banding techniques. Hum Genet 31: 177-196.

Dejmek J, Vojtassak J, Malova J (1992): Cytogenetic analysis of 1508 spontaneous abortions originating from south Slovakia. Eur J obstet Gynecol Reprod Biol 46:129-136.

Eiben B, Bartels I, Bahr-Porsch S, Borgmann S, Gatz G, Gellert G, Goebel R, Hammans W, Hentemann M, Osmers R, Rauskolb R, Hansmann I (1990): Cytogenetic analysis of 750 spontaneous abortions with the direct preparation method of chorionic villi and its implications for studying genetic causes of pregnancy wastage. Am J Hum Genet 47: 656-663.

Eiben B, Borgmann S, Schubbe I, Hansmann I (1987): A cytogenetic study directly from chorionic villi of 140 spontaneous abortions. Hum Genet 77(2): 137-141

French FE, Bierman JM (1967): Probabilities of fetal mortality. Publ Hlth Rep 77: 835-847.

Gardo S, Bajnoczky K (1992): Cytogenetic analysis of spontaneous abortions with direct analysis of chorionic villi. Eur J Obstet Gynecol Reprod Biol 47: 117-120.

Griffin DK, Millie EA, Redline RW, Hassold TJ, Zaragoza MV (1997): Cytogenetic analysis of spontaneous abortions: Comparison of techniques and assesment of the incidence of confined placental mosaicism. Am J Med Genet 72: 297-301.

Guerneri S, Bettio A, Simoni G, Brambati B, Lanzani A, Fraccaro M (1987): Prevalence and distribution of chromosome abnormalities in a sample of first trimester internal abortions. Hum Reprod 2: 735-739.

Hacker N, Moore G (1998): Essentials of Obstetrics and Gynecology, pp. 477-86. (WB Saunders Company U.S.A.)

Hassold T, Chen N, Funkhouser J, Jooss T, Manuel B, Matsuura J, Matsuyama A, Wilson C, Yamane JA, Jacobs PA (1980): A cytogenetic study of 1000 spontaneous abortions. Ann Hum Genet 44: 151-178.

Jacobs PA, Hassold TJ (1987): Chromosome abnormalities: origin and etiology in abortions and livebirths. In Vogel F, Sperling K (eds). Human Genetics: proceedings of the 7^th International Congress, Berlin 1986. Springer, Berlin: 233-244.

Kajii T, Ferrier A, Niikawa N, Takahara H, Ohama K, Avirachan S (1980): Anatomic and chromosomal anomalies in 639 spontaneous abortuses. Hum Genet 55:87-98.

Kline J, Stein Z (1985): Very early pregnancy. In Dixon RL (ed) Reproductive Toxicology. Raven, New York: 29-50.

Morton NE, Jacobs PA, Hassold T, Wu D (1988): Maternal age in trisomy. Ann Hum Genet 52: 227-235.

Ober C (1995): Current topic: HLA and reproduction: lessons from studies in the Hutterites. Placenta 16(7): 569-77.

Ohno M, Maeda T, Matsunobu A (1991): A cytogenetic study of spontaneous abortions with direct analysis of chorionic villi. Obstet Gynecol 77:394-398.

Rossant J, Joyner AZ (1989): Towards a molecular-genetic analysis of mammalian development. Trends Genet 5:277-283.

Shaffer LG, McCaskill C, Adkins K, Hassold TJ (1998): Systematic search for uniparental disomy in early fetal losses: the results and a review of the literature. Am J Med Genetics 79(5) : 366-72.

Songster G, Sun L, Chang S, Cheung S (1992): Chromosome analysis in spontaneous pregnancy loss: Use of placental villus mesodermal core cell cultures. Am J Med Genet 42: 785-788.

Szulman AE, Surti U (1978): The syndromes of hydatidiform mole. I. Cytogenetic and Morphologic Correlations. Am J Obstet Gynecol 131 (6): 665-71.

Warburton D, Stein Z, Kline J, Susser, M (1980): Chromosome abnormalities in spontaneous abortion. Data from the New York City study. In Porter IH, Hook EB (eds): Human Embryonic and Fetal Death. Academic press, New York and London : 261-287.

Wilcox AJ, Weinberg CR, O’Connor JF, Baird DD, Schlatterer JP, Cant RE, Armstrong EG, Nisula BC (1988): Incidence of early loss of pregnancy. N Engl J Med 319 (4): 189-194.

Table 1

A comparison of the number of karyotypes detected by the PIC and STC methods, respectively

Table 2

A list of the double and multiple trisomies detected in our study

Table 3

A comparison of the number of six major trisomies detected with the PIC and STC method respectively

Table 4

Sex ratios from studies using the tissue culture method, compared to the sex ratio for cases cultured by the STC method in our study

Table 5

Sex ratios from studies using the direct preparation of metaphases from chorionic villi compared with the sex ratio for samples processed by the PIC method in our study