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International Journal of Clinical and Experimental Pathology logoLink to International Journal of Clinical and Experimental Pathology
. 2014 Jul 15;7(8):4704–4719.

STR DNA genotyping of hydatidiform moles in South China

Xing-Zheng Zheng 1, Pei Hui 2, Bin Chang 3, Zhi-Bin Gao 4, Yan Li 1, Bing-Quan Wu 1, Bo Zhang 1
PMCID: PMC4152032  PMID: 25197342

Abstract

Objective: To evacuate whether short-tandem-repeat (STR) DNA genotyping is effective for diagnostic measure to precisely classify hydatidiform moles. Methods: 150 cases were selected based on histologic features that were previously diagnosed or suspected molar pregnancy. All sections were stained with hematoxylin as a quality control method, and guided the microscopic dissection. DNA was extracted from dissected chorionic villi and paired maternal endometrial FFPE tissue sections. Then, STR DNA genotyping was performed by AmpFlSTR® SinofilerTM PCR Amplification system (Applied Biosystems, Inc). Data collection and analysis were carried out using GeneMapper® ID-X version 1.2 (Applied Biosystems, Inc). Results: DNA genotyping was informative in all cases, leading to identification of 129 cases with abnormal genotype, including 95 complete and 34 partial moles, except 4 cases failed in PCR. Among 95 complete moles, 92 cases were monospermic and three were dispermic. Among 34 partial moles, 32 were dispermic and 2 were monospermic. The remaining 17 cases were balanced biallelic gestations. Conclusion: STR DNA genotyping is effective for diagnostic measure to precisely classify hydatidiform moles. And in the absence of laser capture microdissection (LCM), hematoxylin staining plus manual dissection under microscopic guided is a more economic and practical method.

Keywords: Short-tandem-repeat (STR), DNA genotyping, diagnosis, hydatidiform mole, non-molar gestation

Introduction

Hydatidiform mole (HM) is an abnormal pregnancy with nonneoplastic proliferation of trophoblasts [1]. It can be divided into two separate syndromes based on morphologic, genetic and clinical factors [2]. The complete hydatidiform mole (CHM) is a diploid androgenetic conceptus with generalized villous trophoblastic hyperplasia and hydatidiform villous swelling in the absence of an ascertainable fetus. The partial hydatidiform mole (PHM) is a diandric triploid conceptus with focal trophoblastic hyperplasia and focal hydatidiform villous swelling, and with a demonstrable fetus. It is clinically important to distinguish a hydatidiform mole from a non-molar hydropic abortus, primarily because of the associated risk of post-molar gestational trophoblastic neoplasia and subsequent clinical follow-up and management of the patient [3]. Furthermore, because a complete mole has a much higher risk of progression to gestational trophoblastic neoplasia (18-29%) than a partial mole (1.0-5.6%), it is necessary to subclassify for hydatidiform moles [4,5]. However, histological evaluation of complete hydatidiform mole (especial early CHM), partial hydatidiform mole, digynic gestation and non-molar hydropic abortion is very difficult and easily mistaken [6]. In the past, many ancillary studies, including DNA ploidy analysis, p57 immunohistochemistry, chromosomal enumeration by fluorescent in situ hybridization (FISH), and DNA short-tandem-repeat (STR) genotyping have been developed [7-9]. Most of them were based on the genetic level of hydatidiform moles specific parental chromosomal complements [10].

At the genotype level, most CHMs are androgenetic, containing two sets of paternal chromosomes, with either 46, XX diploid karyotype (monospermic or homozygous, 80%), or 46, XX or XY karyotype (dispermic or heterozygous, 20%) [11]. In rare cases, CHMs are diploid, with both a maternal and a paternal chromosome complement (Biparental Complement Hydatidiform Mole, BiCHM) [12]. BiCHM is a recurrent complete mole with strong familial tendency, and some studies reported that NLRP7 gene might be the fundamental genetic of BiCHM [12,13]. PHMs are triploid, containing one maternal and two paternal sets of chromosomes, with XXX or XXY triploid karyotype with a diandric, monogynic genome arising from fertilization of a haploid egg by either two spermatozoa (dispermic or heterozygous, 90%) or one spermatozoon with duplication (monospermic or homozygous, 10%) [11]. Because of the earlier clinical detection and curettage of abnormal pregnancies, the histopathological features that are often used to distinguish complete moles, partial moles, and non-molar abortions are more subtle and less readily identifiable, leading to increasing difficulties in the proper subclassification of HMs [4,14]. In daily clinical practice, under-diagnosis of complete mole as partial mole (or non-molar pregnancy), or over-diagnosis of non-molar pregnancy as partial mole (or complete mole) is often encountered [15,16]. Along with the people aware of that the different subtypes have different clinical treatments; the accurate subclassification diagnosis is getting more and more attention in hydatidiform moles. Now, a variety of molecular methods targeting the genetic alterations of hydatidiform moles have been explored to improve diagnostic accuracy [3]. We recently had established a STR analysis platform for DNA genotyping in the diagnosis of molar pregnancy. Our objective was to estimate whether molecular genotyping is effective for diagnostic measure to precisely classify hydatidiform moles.

Materials and methods

Patients and histological evaluation

A total of 150 abortion specimens were selected from February 2009 to March 2014 in department of pathology of YuRao People’s Hospital (ZheJiang Province, China). All cases raised the possibility of molar pregnancy based on histology (Table 1), with diagnostic terms including “favor molar pregnancy”, “consistent with molar pregnancy”, “suggestive of or suspicious for molar pregnancy”, and “rule out molar pregnancy”. All cases had some degree of suspicion for molar gestation by the primary pathologist on the basis of morphologic and/or clinical findings. There were 91 cases that had performed P57 immunohistochemistry mentioned in retrospective data. This study was approved by the institutional review board (Human Investigation Committee of YuRao People’s Hospital).

Table 1.

Clinicopathologic features and Genotyping Diagosis of 150 cases

Patient Age (y) Pathologic Diagnosis p57kip2 Genotyping Diagnosis
1 51 Consistent with HM POS. DPM
2 21 Consistent with HM ND. MCM
3 27 Consistent with HM ND. MCM
4 23 Consistent with HM NEG. MCM
5 47 Suggestive of HM POS. Non-molar gestation
6 27 Consistent with HM NEG. MCM
7 20 Consistent with HM ND. MCM
8 27 Suggestive of HM ND. MCM
9 24 Consistent with HM ND. MCM
10 24 Suggestive of HM ND. DPM
11 27 Consistent with HM ND. MCM
12 22 Consistent with HM ND. MCM
13 30 Consistent with HM NEG. MCM
14 22 Consistent with HM POS. DPM
15 28 Suggestive of HM NEG. MCM
16 28 Consistent with HM ND. MCM
17 18 Consistent with HM NEG. MCM
18 22 Consistent with HM POS. DPM
19 25 Consistent with HM ND. MCM
20 30 Consistent with HM NEG. MCM
21 19 Consistent with HM Focal POS. MCM
22 21 Suggestive of HM NEG. MCM
23 41 Consistent with HM NEG. MCM
24 29 Suggestive of PHM ND. MCM
25 31 Suggestive of HM NEG. MCM
26 25 Suggestive of HM POS. DPM
27 26 Consistent with HM NEG. MCM
28 26 Consistent with HM ND. MCM
29 47 Consistent with HM NEG. MCM
30 20 Consistent with HM ND. MCM
31 28 Consistent with HM ND. MCM
32 44 Consistent with HM NEG. MCM
33 22 Consistent with HM NEG. MCM
34 28 Consistent with HM ND. MCM
35 26 Consistent with HM ND. Failed detection
36 20 Consistent with HM ND. MCM
37 21 Consistent with HM NEG. MCM
38 26 Suggestive of HM NEG. MCM
39 29 Suggestive of HM ND. Failed detection
40 19 Consistent with HM NEG. MCM
41 30 Suggestive of HM NEG. MCM
42 21 Consistent with HM ND. MCM
43 41 Consistent with HM ND. MCM
44 41 Suggestive of HM NEG. MCM
45 32 Favor HM ND. MCM
46 25 Suggestive of HM NEG. MCM
47 34 Favor HM NEG. MCM
48 20 Favor HM NEG. MCM
49 25 Consistent with HM ND. MCM
50 25 Suggestive of HM ND. MCM
51 23 Suggestive of HM NEG. MCM
52 47 Rule out HM POS. Non-molar gestation
53 21 Rule out HM NEG. MCM
54 48 Rule out HM ND. Failed detection
55 26 Suggestive of HM NEG. DCM
56 28 Suggestive of HM NEG. MCM
57 43 Suggestive of HM NEG. MCM
58 27 Consistent with HM NEG. MCM
59 20 Rule out HM ND. MCM
60 28 Consistent with HM NEG. MCM
61 22 Suggestive of HM NEG. MCM
62 22 Suggestive of HM ND. MCM
63 46 Consistent with HM ND. MCM
64 37 Suggestive of HM NEG. MCM
65 33 Rule out HM POS. Non-molar gestation
66 28 Rule out HM POS. DPM
67 29 Suggestive of CHM Focal POS. DPM
68 34 Rule out HM Focal POS. DPM
69 26 Suggestive of PHM POS. DPM
70 28 Rule out HM NEG. MCM
71 26 Suggestive of CHM Focal POS. DPM
72 30 Suggestive of PHM POS. DPM
73 24 Suggestive of PHM POS. Non-molar gestation
74 35 Suggestive of HM POS. DPM
75 30 Suggestive of HM Focal POS. DPM
76 28 Rule out HM POS. Non-molar gestation
77 26 Rule out HM ND. Non-molar gestation
78 29 Suggestive of PHM ND. Non-molar gestation
79 27 Suggestive of HM ND. Non-molar gestation
80 22 Suggestive of HM ND. Non-molar gestation
81 34 Consistent with PHM POS. DPM
82 26 Suggestive of PHM POS. MPM
83 23 Consistent with PHM POS. DPM
84 31 Consistent with PHM POS. DPM
85 25 Consistent with PHM POS. Non-molar gestation
86 34 Suggestive of PHM ND. DPM
87 26 Consistent with PHM POS. DPM
88 28 Consistent with PHM POS. DPM
89 21 Consistent with PHM NEG. DPM
90 29 Suggestive of PHM ND. DPM
91 38 Consistent with PHM POS. DPM
92 26 Rule out CHM ND. DPM
93 32 Suggestive of PHM ND. DPM
94 39 Rule out HM NEG. DPM
95 33 Suggestive of CHM NEG. MCM
96 44 Suggestive of CHM ND. Non-molar gestation
97 31 Consistent with CHM NEG. MCM
98 22 Consistent with CHM NEG. MCM
99 34 Consistent with CHM NEG. MCM
100 24 Rule out HM Focal POS. DPM
101 29 Consistent with CHM NEG. MCM
102 25 Consistent with CHM ND. MCM
103 32 Consistent with CHM ND. DCM
104 20 Consistent with CHM ND. MCM
105 22 Consistent with CHM NEG. MCM
106 25 Suggestive of CHM ND. DCM
107 22 Consistent with CHM ND. MCM
108 34 Consistent with CHM ND. MCM
109 32 Consistent with CHM ND. MCM
110 28 Suggestive of CHM ND. MCM
111 33 Consistent with CHM NEG. MCM
112 29 Consistent with PHM Focal POS. MPM
113 31 Consistent with CHM NEG. MCM
114 27 Suggestive of CHM ND. MCM
115 32 Consistent with CHM ND. Failed detection
116 26 Suggestive of PHM POS. DPM
117 23 Consistent with PHM POS. DPM
118 18 Consistent with PHM POS. DPM
119 27 Consistent with CHM POS. DPM
120 30 Consistent with CHM NEG. MCM
121 22 Consistent with CHM NEG. MCM
122 24 Consistent with CHM NEG. MCM
123 27 Consistent with CHM NEG. MCM
124 27 Consistent with CHM ND. MCM
125 32 Consistent with CHM NEG. MCM
126 20 Consistent with CHM NEG. MCM
127 32 Consistent with CHM ND. MCM
128 23 Consistent with CHM NEG. MCM
129 21 Suspicious for Early CHM Focal POS. MCM
130 22 Suggestive of CHM NEG. MCM
131 31 Consistent with CHM ND. MCM
132 29 Suggestive of CHM NEG. MCM
133 32 Consistent with CHM ND. MCM
134 32 Consistent with CHM NEG. MCM
135 20 Consistent with CHM NEG. MCM
136 28 Suggestive of CHM ND. MCM
137 38 Consistent with CHM NEG. MCM
138 27 Consistent with CHM ND. MCM
139 29 Consistent with CHM NEG. MCM
140 26 Consistent with CHM NEG. MCM
141 24 Suggestive of CHM POS. DPM
142 23 Rule out Early CHM POS. Non-molar gestation
143 24 Rule out HM ND. DPM
144 26 Suggestive of HM POS. Non-molar gestation
145 24 Suggestive of HM NEG. MCM
146 27 Rule out Early CHM ND. Non-molar gestation
147 23 Rule out HM POS. Non-molar gestation
148 22 Rule out HM ND. MCM
149 26 Suggestive of HM POS. Non-molar gestation
150 28 Rule out HM POS. Non-molar gestation
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HM, hydatidiform mole; CHM, complete hydatidiform mole; PHM, partial hydatidiform mole; MCM, monospermic CHM; DCM, dispermic CHM; DPM, dispermic PHM; MPM, monospermic PHM; POS, positive; NEG, negative; ND, not done.

Referring to Buza N’s literature [15], we selected some important parameters to systematically assess, including villous hydrops, maximum size of chorionic villi, villous shape and contour, villous populations, trophoblastic pseudoinclusions, cistern formation, trophoblast hyperplasia, nucleated fetal red blood cells, and other fetal tissues. Each case has been reviewed independently by two gynecologic pathologists. At the same time, we eliminated a few cases which were absence/rare of decidua or rare, finally, 150 cases were selected for STR analyses.

Molecular genotyping detection

Five serial sections 10 micrometers thick were cut from formalin-fixed-paraffin-embedded (FFPE) tissue blocks, the middle section was stained with hematoxylin and eosin to verify the distribution of villous and decidua tissue. In order to isolate pure populations, the remaining four sections were stained with hematoxylin (Dyeing time less than 30 seconds) before the microscopic dissection. Paired tissue samples of chorionic villi and decidua were subjected to DNA extraction by Hydrothermal Pressure (Pressure Cooking) coupled with chaotropic salt column purification method [17,18]. DNA was quantified by spectrophotometric absorbance at 260nm using the NanoDrop apparatus (Thermo Scientific Inc.; Wilmington, DE). The quality of the extracted DNA was evaluated by reading the optical density ratio of 260/280. Genotyping was performed with an AmpFlSTR® Sinofiler™ PCR Amplification Kit (Sinofiler kit) (Applied Biosystems, Inc., Foster City, CA). The reaction consists of a short tandem repeat multiplex polymerase chain reaction (PCR) assay that amplifies 15 different autosomal STR loci (D8S1179, D21S11, D7S820, CSF1PO, D3S1358, D5S818, D13S317, D16S539, D2S1338, D19S433, vWA, D12S391, D18S51, D6S1043, FGA) and the sex-determining marker(Amelogenin) in a single PCR reaction. The producing short amplicons are ranging from 100 to 350 bp. Genomic DNA of 20 to 40 ng was amplified in a 25-microliter reaction containing 10.5 microliters of AmpFlSTR reaction mix, 5.5 microliters of AmpFlSTR® Sinofiler™ primer mix, and 0.5 microliters of AmpliTaq Gold DNA polymerase. The PCR reaction consisted of 11 minutes at 95°C, followed by 28 cycles of 94°C for 1 minute, 59°C for 1 minute, and 72°C for 1 minute, finished by 60°C for 60 minutes. One microliter of the PCR product was mixed with 8.7 microliters of Hi-Di and 0.3-microliter sizing marker (GeneScan-600LIZ; Applied Biosystems, Inc.), followed by capillary electrophoresis on an ABI3500 platform. Data collection and analysis were performed using GeneMapper® ID-X version 1.2 (Applied Biosystems, Inc).

Molecular diagnostic criteria [3]: 1) A molecular diagnosis of complete hydatidiform mole was made when the genotyping profiles of the villous tissue demonstrated exclusively paternal alleles of either monospermic (homozygous paternal alleles) or dispermic (heterozygous paternal alleles) patterns. 2) Dispermic (diandric-monogynic genome) partial hydatidiform mole was diagnosed when the genotyping profiles of the villous tissue showed two distinct paternal alleles in at least two loci but other alleles consist of a duplicate quantity homozygous paternal and one maternal allele. And, monospermic (monospermic duplicate and monogynic genome) partial hydatidiform mole demonstrated homozygous paternal alleles in duplicate quantity, in addition to the presence of one maternal allele in the villous tissue. 3) When the genotyping profiles of the villous tissue showed three alleles in each locus and, two of the three alleles of the villi matched the two maternal alleles of the gestational endometrium, triploid digynic-monoandric gestation was diagnosed. 4) Non-molar gestations, including hydropic abortus, showed balanced biallelic profiles of both paternal and maternal origins in the villous tissue. 5) If the genotyping profiles of the most villous tissue were simiar to nonmolar gestation except only one locus was three alleles or one allele, trisomy or monosomy syndrome diagnosis was made.

Results

DNA genotyping was informative in all cases (Table 1), of which 146 cases were succeeded genotype, including 129 hydatidiform moles (95 complete and 34 partial moles) and 17 non-molar gestations (Figure 1). Among 95 complete moles, 92 cases were monospermic (Figure 2) and three were dispermic. Among 34 partial moles, 32 were dispermic (Figure 3) and 2 were monospermic (Figure 4). 79 cases with histologic diagnostic terms HMs (including consistent with HM, suggestive of HM, rule out HM) were accurate sub-classified, including 55 monospermic complete moles, 12 dispermic partial moles, one dispermic complete moles and 11 non-molar gestations. 17 cases which diagnosed HMs and PHMs/CHMs by their histologic changes were confirmed non-molar hydropic abortion with DNA genotyping (Figure 5A-C). Furthermore, one PHM and 5 CHMs which diagnosed by their histologic changes were precise diagnosed as a monospermic complete mole and 5 dispermic partial moles. We founded 93 cases which had performed p57kip2 immunohistochemistry from retrospective study (Table 1). 56 cases with p57kip2 negative, including 54 cases of CHMs (including 53 MCMs and one DCM) and 2 PHMs. Among 39 cases of p57kip2 positive samples, 26 cases were PHMs (including 24 DPMs and two MPMs), 11 cases were non-molar gestation, and 2 cases were CHMs (Table 2).

Figure 1.

Figure 1

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Genetic profiles of a non-molar gestation demonstrating balanced biallelic profiles of both paternal and maternal origins in the villous tissue (top) similar to the maternal endometrium (bottom).

Figure 2.

Figure 2

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Genetic profiles of a monospermic complete hydatidiform mole. It is demonstrating exclusively paternal alleles in the villous tissue (top). Normal biallelic profiles seen in the maternal endometrium (bottom).

Figure 3.

Figure 3

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Genetic profiles of a dispermic partial hydatidiform mole. It is showing dispermic paternal alleles (two loci with heterozygous paternal alleles and three loci with homozygous paternal alleles in duplicate quantity) , in addition to the presence of one maternal allele (top). Normal biallelic profiles seen in the maternal endometrium (bottom).

Figure 4.

Figure 4

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Genetic profiles of a monospermic partial hydatidiform mole showing demonstrated homozygous paternal alleles in duplicate quantity, in addition to the presence of one maternal allele in the villous tissue (top).Normal biallelic profiles seen in the maternal endometrium (bottom).

Figure 5.

Figure 5

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Non-molar gestation. It had been wrongly diagnosed as a PHM by its morphologic features including enlarged admixed with normal sized villi, villous stromal edema with cistern formation, focal trophoblastic hyperplasia (A, B). (C) Genetic profiles of a non-molar gestation demonstrating balanced biallelic profiles of both paternal and maternal origins in the villous tissue (top) similar to the maternal endometrium (bottom).

Table 2.

Genotyping diagnosis and p57kip2 immunohistochemistry of 95 cases

Genotyping Diagnosis p57kip2 immunohistochemistry
- +
CHM
    MCM 53 2
    DCM 1 0
PHM
    DPM 2 24
    MPM 0 2
Non-molar gestation 0 11
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Discussion

Hydatidiform moles are common diagnostic entities in the daily practice of gynecological pathology. It is an abnormal pregnancy with nonneoplastic proliferation of trophoblasts and occurs in about 1 in 1000-1500 pregnancies in Western countries and is somewhat more frequent in Latin America, Southeast Asia and the Middle East [19,20]. In China, the reported incidences of HMs vary from 1 to 8.83 in every 1000 pregnancies, with the highest incidence being in the province of Zhejiang [21]. Then, Prof. Shi et al reported an incidence of HMs was about 2.5 in every 1000 pregnancies from 143 hospitals in 1990s [22]. As is well-known there are some limitation in HMs pathologic diagnosis, the exact frequency is not known. Although the common form of this disorder is sporadic, 1-6% of patients with a prior mole will have a second mole, and 10-20% will have a second non-molar reproductive wastage, most commonly a spontaneous abortion [23]. It is clinically important to distinguish a hydatidiform mole from a non-molar hydropic abortus, primarily because of the associated risk of post-molar gestational trophoblastic neoplasia and subsequent clinical follow up and management of the patient. Accurate subclassification of hydatidiform moles is also important, as a complete mole has a much higher risk of progression to gestational trophoblastic neoplasia (18-29%) than a partial mole (1.0-5.6%) [4,5]. Although they occur infrequently, gestational trophoblastic tumors are important to recognize because of their varying clinical behaviors and overlapping histological features with common uterine malignancies.

Histologic changes of early complete molar pregnancy included enlarged chorionic villi with polypoid configurations, cellular myxoid stroma, and mild nonpolar hyperplasia of trophoblasts. Histologic features suspicious for partial molar pregnancy included the presence of fetal parts, enlarged admixed with normal sized villi, villous stromal edema with cistern formation, villi with irregular (scalloping) contours and trophoblast inclusions, and nonpolar hyperplasia of syncytiotrophoblast. In approximately 50% of complete moles and 74% of partial moles, the pathologic diagnoses are incorrectly made in the absence of ancillary studies, even in a gynecologic specialty practice setting [16,24]. Our trial showed 79 cases with histologic diagnostic terms HM, including “suggestive of or suspicious for molar pregnancy” and “rule out molar pregnancy”, were obtained accurate subclassification by PCR-based short tandem repeat DNA genotyping. And 17 cases which diagnosed HMs and CHMs/PHMs by their histologic changes, were confirmed non-molar hydropic abortion by DNA genotyping. So, in order to improve the accuracy and perform subclassification of HMs, a variety of ancillary techniques can aid in the diagnosis. These include karyotyping, DNA ploidy flow cytometry, chromosomal enumeration by fluorescent in situ hybridization (FISH), and PCR-based short tandem repeat DNA genotyping [7-9].

Although conventional karyotyping is the most accurate chromosomal enumeration method that may be used to confirm the presence of triploidy in a partial mole or diploidy in a complete mole, it cannot specifically ascertain the parental origin of chromosomal contribution to the gestational tissue [25]. DNA ploidy analysis by flow cytometry is frequently used for the separation of a partial mole from a complete mole or a diploid non-molar hydropic abortus by a demonstration of triploidy [26]. However, it is not useful in the distinction between a complete mole and a non-molar hydropic abortus. Furthermore, DNA ploidy analysis cannot distinguish a digynic-monoandric non-molar gestation from a true diandric-monogynic partial mole. In addition, the use of flow cytometry for FFPE material causes not only the problems of tissue contamination, but also cultural artifacts and random or inadequate sampling, so, often increases therefore the specificity additionally by the use of a citrate buffer and RNAse digestion [27,28]. So, flow cytometry ploidy analysis using FFPE tissue is frequently plagued with technical difficulties and interpretation errors, resulting in significant misclassification of ploidy and misdiagnosis of hydatidiform mole [29]. Interphase FISH can be used for the determination of the number of haploid chromosome sets using both fresh and FFPE tissue samples. But, similar to ploidy analysis, it cannot distinguish a diploid complete mole from a non-molar hydropic abortus and is unable to separate a true diandric-monogynic partial mole from a digynic-monoandric non-molar gestation [8,30]. Because the above methods have their limitations, in particular, they cannot specifically ascertain the parental origin of chromosomal contribution to the gestational tissue, we need to combine histological morphology and clinical information to analyze.

With IHC markers such as p57kip2 used, to some extent, the accuracy rate HMs diagnosis has improved. P57kip2 expression has been found to be useful in the distinction of CHMs (including early forms) from PHMs and NMs; however, the latter two entities cannot be distinguished from one another because of shared (retained) p57kip2 expression patterns. CHMs, including the early forms, which lack a maternal genetic contribution, have absent (or very limited) p57kip2 expression in villous stromal cells and cytotrophoblast, but positive in intervillous intermediate trophoblast, villous endothelial cells, and gestational endometrium [31,32]. In contrast, both PHMs and NMs (including those with abnormal villous morphology), contain a maternal chromosomal complement and exhibit diffuse p57kip2 expression in these cell types, show strong nuclear p57kip2 expression in cytotrophoblast, intermediate trophoblast, villous stromal cells, and decidual stromal cells [31,33]. In our retrospective information, there were about 96.4% (54/56) showed p57kip2 immunohistochemistry negative expression in CHMs. A weak nuclear staining was showed in 2 CHMs, probably, that might be in part because of p57 gene incompletely inactive. The cases of p57kip2 immunohistochemistry positive expression included 26 PHMs and 11 non-molar gestations. Two DPMs showed p57kip2 immunohistochemistry negative expression, the main reason maybe lie in the inadequate of antigen exposure. Overall, p57kip2 immunohistochemistry can aid in the diagnosis. But, it cannot differentiate PHM from its mimics that contain maternal genetic material (hydropic abortions, trisomies). A recent study believed that there was different biological behavior between heterozygous and homozygous complete moles, the former have a more aggressive than the latter [34]. Including p57kip2 immunohistochemistry and DNA ploidy analysis, these methods cannot distinguish them. Until a few years ago, some studies have demonstrated the value of STR genotyping, for distinguishing HM from non-molar gestations and for subtyping HMs as CHM and PHM [9].

STR genotyping allows for determination of both ploidy and the maternal/ paternal contributions of chromosome complements. Thus, it can distinguish these entities by discerning androgenetic diploidy, diandric triploidy, and biparental diploidy to diagnose CHMs, PHMs, and NMs, respectively. STR is highly prevalent noncoding repetitive DNA sequences of 2 to 7 nucleotides in the human genome and are genetically stable [35]. STR polymorphism denotes that a STR locus differs in the number of repeats between individuals. By identification of the number of STR at specific loci, a genetic profile of an individual or a cell can be ascertained to distinguish one from another. STR polymorphism analysis of gestational tissue in comparison with corresponding maternal tissue offers a determination of parental genomic contribution and therefore can diagnose and sub-classify hydatidiform moles at the genetic level [9,11,36]. In this study, we evaluated 146 products of conception at the genetic level. 129 cases with abnormal genotype were identified, including 95 complete and 34 partial moles. Among 95 complete moles, 92 cases were monospermic and three were dispermic. Among 33 partial moles, 28 were dispermic and 5 were monospermic. It is important to note that 79 cases with histologic diagnostic terms HMs were accurate sub-classified, and 17 cases which diagnosed HMs and PHM/CHM by their histologic changes were confirmed non-molar hydropic abortion with DNA genotyping. 79 cases with histologic diagnostic terms HMs (including consistent with HM, suggestive of HM, rule out HM) were sub-classified into 55 monospermic complete moles, 12 dispermic partial moles, one dispermic complete moles and 11 non-molar gestations. Furthermore, one PHMs and 5 CHMs which diagnosed by their histologic changes were precise diagnosed as a monospermic complete mole and 5 dispermic partial moles. So, STR DNA genotyping is a practical and highly accurate method for the subclassification of hydatidiform moles.

STR genotyping for molar pregnancy assay resembles a conventional diagnostic molecular procedure, including manual tissue dissection, DNA extraction, a STR multiplex PCR reaction, capillary electrophoresis, and data analysis. The first step is to dissect the villous and maternal tissue as far as possible; it is the key to molecular diagnosis of HMs. In most tissue samples of product of conception, well-defined areas of chorionic villi and maternal endometrium are easily recognized in serial tissue sections and can be safely individually dissected into separate test tubes [3]. But, we had been aware of that the position and size of the villous and maternal tissue in each section would be differences, and it was hard to avoid tissue cross-contamination. Therefore, it is not accurate only by one HE section evaluates the distribution of villous and decidua tissue. In order to isolate pure populations, the remaining sections were stained with hematoxylin (Dyeing time less than 30 seconds) before the microscopic dissection. Furthermore, this process does not affect the follow-up experiment (data not shown). Of course, an absolutely pure isolation of villous tissue is generally impossible, as maternal blood and endometrial tissue or cells may be intimately admixed with chorionic villous tissue [3,11]. In our experiment, we used a novel Hydrothermal Pressure (Pressure Cooking) coupled with chaotropic salt column purification method for DNA extraction. Under the prerequisite of guaranteeing DNA quality, this method does not only short DNA extraction time, but also greatly reduce the cost of reagent. Genotyping was performed with an AmpFlSTR® Sinofiler™ PCR Amplification Kit. The reaction consists of a short tandem repeat multiplex polymerase chain reaction (PCR) assay that amplifies 15 different autosomal STR loci and the sex-determining marker (Amelogenin) in a single PCR reaction. This kit employs the same primer sequences as used in the previous AmpFlSTR® kits with the exception of D6S1043 and D12S391. Degenerate primers for the loci D8S1179, vWA, and D16S539 were added to the AmpFlSTR® SinofilerTM Primer Set to address mutations in the primer binding sites. The producing short amplicons are ranging from 100 to 350 bp, suitable for FFPE tissue samples. The data were derived and then analyzed by GeneMapper® ID-X version 1.2 after capillary electrophoresis. Interpretation of genotyping data is generally straightforward when the genotyping profile of the pure villous tissue is compared with that of the maternal tissue. The detailed interpretation can refer to “molecular diagnostic criteria” (the part of “Methods”). A few potential pitfalls cannot be ignored in the genotypic diagnosis of a small subset of complete mole of biparental origin, as both the paternal and the maternal genomes are present in the villus and decidua tissue, DNA genotyping is not helpful [3,13]. In addition, a gestation derived from an egg donor pregnancy is confusing to the genotypic diagnosis. Because a donor egg will present STR alleles that may simulate a dispermic complete mole, DNA genotyping cannot distinguish an egg donor pregnancy from a true dispermic complete mole [3]. And, hydatidiform moles arising from a twin gestation may also potentially complicate analysis [3,37]. Clinical information (recurrent mole, egg donor recipient, and twin gestation) and careful morphological assessment of the tissue, followed by isolation of pure hydropic villi for genotyping comparison, may resolve such difficult cases [3]. When there is discordance between the genotyping result and the morphology, p57kip2 immunohistochemistry is helpful to identify rare cases of mosaicism, chimerism, or CHM arising from a twin gestation [38,39]. P57kip2 immunohistochemistry and PCR-based STR DNA genotyping are powerful discriminatory markers that can be used to precisely diagnose and subtype both complete and partial hydatidiform moles.

Through this study, we believe that DNA genotyping can be effective for diagnostic measure to precisely classify hydatidiform moles. And in the absence of laser capture microdissection (LCM), hematoxylin staining plus dissection under microscopic guided is a more economic and practical method. In China, the research in hydatidiform moles by DNA genotyping is stills less, not to mention the application for clinical diagnosis. Although our laboratory has performed mature PCR-based STR DNA genotyping platform through a plenty of preclinical validation study, further studies are needed. Combining morphology and p57kip2 immunohistochemistry as well as clinical information, integrating DNA genotyping to the routine diagnostic algorithms of hydatidiform moles, precise diagnose and subtype may be beneficial to clinical follow-up and management of the patient.

Acknowledgements

This work was supported by National Natural Science Foundation of China (grant No. 81260104). We thank Prof. Gang Li (Department of Biochemistry and Molecular Biology, Peking University Health Science Center) and for his help.

Disclosure of conflict of interest

None.

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