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Keywords:

  • congenital anomaly;
  • descriptive study;
  • human embryo;
  • Kyoto Collection;
  • maternal factor

ABSTRACT

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. ACKNOWLEDGMENTS
  9. REFERENCES

Understanding the causes of congenital anomalies is of prime importance to develop management and/or prevention strategies. It is widely accepted that the occurrence of congenital malformations in fetuses and neonates is heavily correlated with maternal genetic makeup and lifestyle. However, very few epidemiologic analyses have been conducted on the embryonic developmental period because of the rarity of data available. Instigated in 1961, the Kyoto Collection of Human Embryos comprises approximately 45 000 specimens of embryos and fetuses. The collection's most unique feature is that most specimens were added to the collection along with epidemiologic information on the respective mothers. This is the first report on the digitization of data from the collection. A total of 22 262 embryonic specimens were selected on the basis of data integrity. Data related to the embryos were then classified according to the following criteria: developmental stage, sampling period, geographical area, maternal determinant, and external malformation. Results indicate that 7.8% of the embryos exhibit external anomalies and 92.2% are without anomalies. The three most common anomalies were nuchal bleb, holoprosencephaly and spina bifida. A special emphasis was placed on the potential association between maternal determinants and embryonic external anomalies, allowing for statistical analyses. The present study provides further evidence that this collection represents a unique source of information to conduct epidemiological analyses, not only to further the understanding of congenital anomalies but also to help establish preventive health guidelines for pregnant women.


INTRODUCTION

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. ACKNOWLEDGMENTS
  9. REFERENCES

Malformation at birth occurs at a rate of 3% (Martin et al. 2008) and in most cases, the causative factors remain unknown. Based on current published reports, the risk factors associated with congenital anomalies are grouped into internal and external influences (Gruenwald 1947). Internal influences primarily consist of chromosomal abnormalities and other genetic disorders, while external factors are related to maternal influences such as diabetes, abusive consumption of alcohol, cigarette smoking and folic acid deficiency; infectious diseases such as rubella, herpes and toxoplasma; drug intake such as antiepileptic, angiotensin-converting enzyme antagonists and sedative drugs; dietary habits such as food additives; and radiation exposure such as radioactivity or electromagnetic waves stemming from frequent cell phone usage (Wyszynski et al. 2005; Baumann et al. 2006; Divan et al. 2008; Safir et al. 2010; Schmidt et al. 2010; Reef et al. 2011). Because external factors are more amenable to modifications, they have been the focus of past and current investigations aiming at developing efficient strategies for the prevention of congenital anomalies. Many studies have explored the causes of congenital anomalies occurring in fetuses at 20–22 weeks of gestation up to 7-day-old infants (Leoncini et al. 2010); however, very few reports can be found on earlier developmental stages. As embryos or fetuses with severe malformation tend to result in spontaneous abortion at a high rate during the early period of pregnancy (Nishimura et al. 1968; Shiota 1991), research using the samples at the early embryonic period should be performed to resolve the relationship between maternal factors and birth defects. However, despite the existence of large collections of human embryos, it has been difficult to clearly identify the maternal–fetal relationship mainly due to the paucity of maternal data associated with the embryo specimens. This has thus hindered statistical analyses on potential correlations.

Large human embryo collections have greatly contributed to the understanding of early human embryonic morphogenesis. The Carnegie Collection, created in 1887, and the Blechschmidt Collection, initiated in 1948, both represent an impressive collection of embryonic models and both have played a significant role in the history of human embryology. Indeed, the two collections house specimens of historical values and feature some of the most striking serial sections of human embryos (Freeman 2003; Carter 2008). These two collections have allowed researchers to generate 3D-reconstructed models obtained from serial sections, and many reports have been published (see review in Yamada and Takakuwa 2012). The Kyoto Collection of Human Embryos (KC) was created in 1961, and represents a more recent compendium in comparison with the Carnegie or Blechschmidt collections. Major features distinguish KC from other collections; first, KC is the largest collection of human embryos in the world, featuring a reported total of 45 337 specimens. Additionally, because the embryos were collected in a random manner, it is accepted that the embryo population in the collection is representative of an unbiased intrauterine population in Japan (Nishimura et al. 1968; Nishimura 1975; Shiota 1991). Second, KC comprises embryo specimens with a large variety of external malformations as reported by previously published studies (Nishimura et al. 1968; Matsunaga and Shiota 1977; Yamada et al. 2004). A third unique feature of KC is that most specimens were collected along with clinical and epidemiological information on the associated pregnancies and the mothers. These data were accumulated in the formats of paper sheets and punch cards, rendering epidemiological and statistical analyses difficult to perform. To address these issues, a project was undertaken to digitize the clinical and epidemiological data gathered in KC and turn them into textual and imaging data. This is the first report on the integrated database established during the project. Here we provide a detailed overview of the epidemiological and clinical data associated with KC, with a special emphasis on potential associations between maternal risk factors and embryonic external malformations.

MATERIALS AND METHODS

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. ACKNOWLEDGMENTS
  9. REFERENCES

Legal considerations and informed consent of the mothers

The scope and purposes of the study were explained and cooperation was requested from the local prefectural branches of the Japan Medical Association. Local branches then informed their respective obstetricians of the nature of the study. In total, approximately 1400 obstetricians agreed to register and participate in the study. Each obstetrician clearly explained the research purposes to pregnant women scheduled to undergo artificial abortion under circumstances authorized by the Maternal Health Protection Law of Japan. With the written informed consent of the mothers, the specimens were then sampled in association with clinical and epidemiological information on both the embryo and the mother.

Specimens in the collection

In a great majority of cases, the embryos housed in KC were derived from pregnancies that were terminated for social reasons during the first trimester of gestation under circumstances authorized by the Maternal Health Protection Law of Japan. As a consequence, most mothers were considered to be generally healthy women. Because the attending obstetricians did not examine the aborted materials, the collection of embryos was not biased by their outcome (normal or abnormal, live or dead), and the embryo collection is thus considered to be representative of the total intrauterine population in Japan. Once the aborted materials were brought to our laboratory, the embryos were measured, staged and examined for gross external abnormalities and signs of intrauterine death under a dissecting microscope. The specimens were classified according to their developmental stages, as defined by Carnegie stages in accordance with the criteria proposed by O'Rahilly and Müller (1987). Further details on KC can be found in previous publications (Nishimura et al. 1968; Nishimura 1975; Shiota 1991; Yamada et al. 2004).

Construction of a Human Embryo Database of the Kyoto Collection (KCDB)

Epidemiological data obtained from participating obstetricians were recorded in the format of individual punch cards. As explained earlier, for several decades, these data have been stored and processed on paper-based material. The project aiming at digitizing the data made available by KC was completed with use of the computer software FileMaker Pro version 11 (FileMaker, Santa Clara, CA, USA), a widely used program facilitating data conversion and statistical analyses. The database was coined as the KCDB (Human Embryo Database of Kyoto Collection). Results accuracy was carefully checked against paper-based master data. Part of the processed data can be accessed from the Kyoto Human Embryo Visualization Project website (http://bird.cac.med.kyoto-u.ac.jp).

Selection criteria for inclusion of specimens into the study

KC contains a total of 45 337 specimens, housed at the Congenital Anomaly Research Center, Kyoto University Graduate School of Medicine. The collection consists of 39 815 embryos and 5522 fetuses. For this study, embryo specimens were selected on the basis of data integrity using the database obtained above. More specifically, the dataset associated with an embryo was considered integral if it contained the following three types of information: (i) date of operation, (ii) registered obstetrician's identification number, and (iii) clinical and morphological observations on the embryo. As a result, 22 262 of 39 815 embryos, that is, 55.9% of the embryos in KC, were included in the present study (Fig. 1).

image

Figure 1. Inclusion criteria of specimens into the study of the 45 337 human specimens (39 815 embryos and 5522 fetuses) comprising the Kyoto Collection. Slightly less than half (22 262 embryos) were included in the study.

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The study was approved by The Committee of Medical Ethics at the Kyoto University Graduate School of Medicine, Kyoto, Japan (E1016).

RESULTS

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. ACKNOWLEDGMENTS
  9. REFERENCES

Embryo distribution based on developmental stages (Carnegie stages)

Embryos were analyzed under a dissecting microscope and grouped into developmental stages (Carnegie stages, CS), in accordance with the criteria proposed by O'Rahilly and Müller (1987), Of the 22 262 specimens initially selected, it was possible to determine the developmental stages of 21 528 embryos, which ranged from CS3 to CS23 (Table 1). As much as 62.4% of the embryos were within CS16 and CS20, with the most prominent population of embryos (16.3%) in stage CS16. In contrast, less than 2% of total embryos fell within developmental stages CS3 and CS12, and nearly 20% of the specimens were classified CS13–CS15. The remaining 16.5% were collected at developmental stages CS21–CS23. When embryos could not be staged because of damage to each sample, they were grouped as “not determined”. In this population, 7.8% of the embryos (i.e. 1744 specimens) exhibited external anomalies against 92.2% (i.e. 20 518 embryos) without anomalies.

Table 1.  Distribution of embryo specimens according to Carnegie stages (n = 22 262)
Carnegie stageNo. embryos (%)With external malformation (%)Without external malformation (%)
31 (0.0)0 (0.0)1 (100.0)
74 (0.0)0 (0.0)4 (100.0)
84 (0.0)0 (0.0)4 (100.0)
93 (0.0)0 (0.0)3 (100.0)
1020 (0.1)3 (15.0)17 (85.0)
1139 (0.2)4 (10.3)35 (89.7)
12274 (1.2)10 (3.6)264 (96.4)
13702 (3.2)51 (7.3)651 (92.7)
142 018 (9.1)75 (3.7)1 943 (96.3)
151 494 (6.7)46 (3.1)1 448 (96.9)
163 499 (15.7)320 (9.1)3 179 (90.9)
172 573 (11.6)227 (8.8)2 346 (91.2)
182 649 (11.9)192 (7.2)2 457 (92.8)
192 275 (10.2)123 (5.4)2 152 (94.6)
202 406 (10.8)251 (10.4)2 155 (89.6)
211 622 (7.3)235 (14.5)1 387 (85.5)
221 081 (4.9)106 (9.8)975 (90.2)
23864 (3.9)50 (5.8)814 (94.2)
Not determined734 (3.3)51 (6.9)683 (93.1)
Total22 262 (100.0)1 744 (7.8)20 518 (92.2)

Embryo distribution based on sampling period

The embryos specimens of KC were sampled over the period 1960–1999, thus spanning four decades (Table 2). The number of embryos collected within each decade was determined and results are presented in Table 2. Over 70% of the embryos were collected in the 1960s and 25.5% in the following decade (i.e. 1970s). In contrast, we determined that less than 5% of the specimens joined KC between 1980 and 1999.

Table 2.  Distribution of embryo specimens according to the collection period (n = 22 262)
DecadeNo. embryos (%)With external malformation (%)Without external malformation (%)
1961–196915 808 (71.0)697 (4.4)15 111 (95.6)
1970–19795 676 (25.5)913 (16.1)4 763 (83.9)
1980–1989742 (3.3)128 (17.3)614 (82.7)
1990–199936 (0.2)6 (16.7)30 (83.3)

Embryo classification based on geographical areas in Japan

Registered obstetricians were assigned an ID number, allowing tracing of the embryos back to their geographical area of collection (Table 3). Embryos were classified according to their geographical districts first, and then to their prefecture. Results showed that the specimens were collected from a total of 21 prefectures in six different districts. As a result, none of the participating physicians were registered within the districts of Hokkaido, Tohoku and Kyushu.

Table 3.  Distribution of embryo specimens based on geographical collection areas in Japan (n = 22 262)
DistrictPrefectureNo. embryos (%)With external malformation (%)Without external malformation (%)
KansaiKyoto1885 (8.5)125 (6.6)1760 (93.4)
Osaka5645 (25.4)375 (6.6)5270 (93.4)
Hyogo2543 (11.4)178 (7.0)2365 (93.0)
Shiga639 (2.9)31 (4.9)608 (95.1)
Nara380 (1.7)31 (8.2)349 (91.8)
Wakayama100 (0.4)6 (6.0)94 (94.0)
TokaiAichi1670 (7.5)112 (6.7)1558 (93.3)
Gifu1011 (4.5)112 (11.1)899 (88.9)
Mie462 (2.1)20 (4.3)442 (95.7)
Shizuoka611 (2.7)33 (5.4)578 (94.6)
KantoTokyo5805 (26.1)552 (9.0)5283 (91.0)
Kanagawa655 (2.9)101 (15.4)554 (84.6)
Saitama514 (2.3)61 (11.9)453 (88.1)
Chiba67 (0.3)13 (19.4)54 (80.6)
HokurikuToyama137 (0.6)17 (12.4)120 (87.6)
Ishikawa10 (0.0)1 (10.0)9 (90.0)
Fukui61 (0.3)4 (6.6)57 (93.4)
Chugoku/ShikokuEhime9 (0.0)0 (0.0)9 (100)
Kagawa43 (0.2)2 (4.7)41 (95.3)
Okayama1 (0.0)0 (0.0)1 (100)
Kochi14 (0.1)0 (0.0)14 (100)

Prevalence of external anomalies in relation to maternal characteristics

Embryos were divided into two groups depending on the presence (n = 1744) or absence (n = 20 518) of external abnormalities. In an effort to examine the potential relationship between maternal determinants and external anomalies, the presence (with external malformation) or absence (without external malformation) of external malformations was enumerated for each nominative maternal characteristic (Table 4). The maternal factors in the study were age, employment status, smoking habits, alcohol consumption, marital status, spontaneous versus artificial abortion, mode of delivery, prior history of pregnancy, infection, radiation exposure during pregnancy and medication intake.

Table 4.  Classification of embryo specimens according to maternal characteristics
Maternal conditionsWith external malformationWithout external malformationTotal
(n = 1 744)(n = 20 518)(n = 22 262)
  • The maternal age is indicated in years, while the other figures are indicated in percentages.

Maternal age (mean ± SD)29.82 ± 6.329.79 ± 6.129.79 ± 6.1
Unemployed68.470.170
Employed31.629.930
SmokingNo81.683.182.9
Yes18.416.917.1
Alcohol consumptionNo67.966.967.1
Yes32.133.132.9
Consanguineous marriageNo98.998.598.6
Yes1.11.51.4
Spontaneous abortionNo78.384.383.9
Yes21.715.716.1
Artificial abortionNo40.63838.2
Yes59.46261.8
Primipara2925.225.5
Multipara7174.874.5
StillbirthNo96.697.197.1
Yes3.42.92.9
InfectionNo92.994.894.7
Yes7.15.25.3
RadiationNo98.69999
Yes1.41.01.0
Drug usageNo77.282.782.3
Yes22.817.317.7

Further analysis of embryonic external malformations

We described earlier that external anomalies were observed in 1744 embryos, that is, 7.8% of total embryos. Each embryo specimen was observed in detail and clinically diagnosed for external malformations. In total, the embryos were classified into 140 groups of diseases in accordance with the International Classification of Diseases (http://www.who.int/classifications/icd/en/), approved by the World Health Organization. When an embryo presented two or more malformations, it was counted multiple times. In this population, there were 1457 embryos with a single malformation, 206 embryos with two malformations, and 81 embryos with three or more malformations. The 30 most common anomalies are listed in Table 5. In some cases, the nature of the malformations could not be defined with confidence. These specimens were considered independently from the embryos with clearly identifiable malformations. The three most common anomalies were identified as nuchal bleb, holoprosencephaly and spina bifida.

Table 5.  Classification of embryos according to their external malformations
MalformationNumber
  1. (susp.), cases that were suspected of abnormality but could not be clearly diagnosed.

  2. CNS, central nervous system.

Nuchal bleb224
Holoprosencephaly201
Upper limb: polydactyly right, preaxial (susp.)109
Myeloschisis/spina bifida95
Upper limb: polydactyly left, preaxial (susp.)81
Upper limb: polydactyly left, postaxial (susp.)78
Hydromyelia (susp.)73
Upper limb: polydactyly bilateral, postaxial (susp.)53
Upper limb: polydactyly right, preaxial50
Myeloschisis/spina bifida (susp.)50
Upper limb: polydactyly left, preaxial49
Other CNS anomalies (susp.)49
Exencephaly46
Other upper limb anomalies46
Branchial arch anomalies45
Cleft lip (isolated) bilateral40
Upper limb: polydactyly bilateral, preaxial36
Upper limb: polydactyly right, preaxial (susp.)34
Upper limb: polydactyly bilateral, preaxial (susp.)33
Lower limb: polydactyly left, postaxial (susp.)31
Holoprosencephaly (susp.)29
Branchial arch anomalies (susp.)24
Ventricular septal defect22
Cleft lip (isolated) median21
Microcephaly (susp.)21
Lower limb: polydactyly bilateral, postaxial (susp.)19
Exencephaly (susp.)17
Other CNS anomalies16
Cleft lip (isolated) left16

DISCUSSION

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. ACKNOWLEDGMENTS
  9. REFERENCES

To the best of our knowledge, this is the first report providing evidence of the association between maternal determinants and embryonic malformations using a large-scale embryo collection. A distinctive feature of KC is the existence of epidemiological and clinical information associated with every single embryo. Using KC, previous studies have focused on embryonic diseases. For example, some authors reported the prevalence of holoprosencephaly (HPE) – a severe but rare type of congenital brain malformation (1/10 000–20 000 at birth) – was very high (1/250 or more) in early human embryonic population (Matsunaga and Shiota 1977), while other authors described the various phenotypic manifestations of HPE, as observed in KC (Yamada et al. 2004). Using 1213 embryos with developmental anomalies, Nishimura et al. (1968) determined that severe congenital anomalies occurred at a higher rate in embryos compared to infants. Similarly, our results indicate that external anomalies were observed in 7.8% of total embryos, which is higher than the prevalence reported in newborns (Martin et al. 2008). The database created by the Japan Association of Obstetricians and Gynecologists indicates that the occurrence of congenital anomalies in Japan has been stable over the past 40 years (Hirahara 2007). This suggests that our data – ranging from the 1960s to the 1990s – can be reliably utilized for descriptive and statistical analysis.

In the present study, data on maternal characteristics and clinical observations of embryos were carefully re-examined, and a new database was constructed. Our results determined that 22 262 of 45 337 specimens have sufficient data integrity to allow for further statistical analyses. Within the selected specimens, a large variety of anomalies were described (Table 5), allowing for a closer analysis of the causes of congenital anomalies. The relationships between the prevalence of external anomalies versus developmental stages, sampling period, and geographical areas in Japan were also described. However, its interpretation is not simple and further detailed analyses will be required to understand their correlation. Many reports have demonstrated the causal relationship between congenital anomalies and maternal factors, such as smoking, alcohol drinking, maternal obesity and diabetes (Miller et al. 2009; Rankin et al. 2010; Alverson et al. 2011; Flenady et al. 2011; Grewal et al. 2011). These reports revealed the association between risk factors and frequency of anomalies. Our aim is to explore the existence of a causal relationship between congenital anomalies and maternal factors by exploitation of large-scale human embryo collections such as KC. The approach is similar to that of elucidating the relationship between neural tube defects and folic acid deficiency (Blencowe et al. 2010).

In recent years, congenital anomalies have been diagnosed prenatally owing to the progress of visualization techniques, such as ultrasonography, magnetic resonance imaging, and X-ray computed tomography. Visualization modalities for embryos and fetuses in the uterus continue to progress in such a way that prenatal diagnosis will be performed not only during the fetal developmental period, but also during the embryonic period. It is important to re-emphasize that the embryo population of KC is representative of an unbiased intrauterine population in Japan (Nishimura et al. 1968; Nishimura 1975; Shiota 1991). This suggests that further clinical and epidemiological analyses using KC will facilitate prenatal diagnosis and assist in prenatal obstetrical monitoring.

Maternal determinants such as alcohol consumption and smoking habits did not permit any detailed analyses, thus there are limits to our retrospective study. However, it is important to note that our approach using an established large collection of human embryos is unique, in that the task of constructing a new collection of human embryos would be both technically and ethically challenging. In 2011, the Japan Ministry of the Environment launched a new project entitled the Japan Environment and Children's Study (http://www.env.go.jp/en/chemi/hs/jecs/) aiming to investigate the impact of environmental chemicals on children's growth and disease prevalence, including the prevalence of congenital malformations. One hundred thousand pregnant women will be recruited for the study and fetal blood will be sampled from the umbilical cord along with the maternal blood and the respective epidemiological data will be recorded. After birth, the recruited children will be followed up to 13 years of age. While the project represents a very large-scale prospective study, the embryonic and fetal periods will not be included in the analysis. Therefore, data such as those presented in our study and collected from embryo and fetus specimens at KC could provide a very useful source of information and would nicely complement this national endeavor.

CONCLUSION

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. ACKNOWLEDGMENTS
  9. REFERENCES

In the present study, an overview of the digitized data gathered from KC was provided. The project aims at the creation of a unique database of human congenital anomaly. We reaffirmed that KC represents a unique tool for epidemiological analyses, and that such a large and varied collection of embryos would be difficult to replicate. While this first report was primarily descriptive, further analyses on the causal relationships between maternal determinants and congenital anomalies are currently being performed to establish preventive health guidelines for pregnant women.

ACKNOWLEDGMENTS

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. ACKNOWLEDGMENTS
  9. REFERENCES

We are immensely grateful to Ms Mari Yamasaki, Dr Lanying Zhao, and Dr Munekazu Komada for their technical support in the construction of the database. We also deeply appreciate Prof. Kohei Shiota, Vice President of Kyoto University for his support and guidance on the project. Grateful acknowledgement is also given to the registered obstetricians who have contributed to the Kyoto Collection of Human Embryos. This research was partially supported by Grants #228073, #238058, and #21790180 from the Japan Society for the Promotion of Science, and the Institute for Bioinformatics Research and Development in the Japan Science and Technology Agency.

REFERENCES

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. ACKNOWLEDGMENTS
  9. REFERENCES