Establishing genetic relationship between a surrogate mother and a child using histological placental blocks: a series of case reports

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INTRODUCTION

Every year, the number of children born via surrogacy increases worldwide¹. This can be explained by the growing interest in starting a family among those with various types of infertility. Improved quality and availability of reproductive medicine, embryology, and pharmacology, along with advancements in clinical laboratory tests and the widespread use of assisted reproductive technology, including in vitro fertilization (IVF), also play a role [1].

Russia actively promotes medical tourism for foreign citizens. Reproductive medicine centers provide services to individuals with infertility from foreign countries where certain techniques, such as surrogacy, are unavailable or illegal [2]. For example, Chinese patients actively use these services. IVF and other types of assisted reproductive technology are not prohibited in China; however, their regulation has not been formalized [3].

There are two types of surrogacy based on the source of the oocyte used for fertilization. Conventional surrogacy uses the surrogate mother’s oocyte, making her the child’s biological mother and providing a genetic relationship. In gestational surrogacy, the implanted embryo is obtained through donor oocyte fertilization; in these cases, the surrogate mother and the child will not be genetically related [4].

In 1995, the D.O. Ott Research Institute of Obstetrics, Gynecology, and Reproductology in St. Petersburg implemented the first gestational surrogacy program², which is the only type of surrogacy approved in Russia. The legal framework for surrogacy was established in 2011–2013^3,4,5.

From the legal standpoint, surrogacy refers to childbearing and delivery under a contract between the surrogate mother who carries the child following donor embryo implantation and the potential parents whose sex cells were used for fertilization.⁵ Cases where a surrogate mother simultaneously acts as an oocyte donor (i.e., the newborn’s biological mother) and then transfers the child to other persons for remuneration under the pretense of a surrogacy program can be classified as illegal. Such cases are considered as a transaction to buy and sell a person (a newborn).⁴

This highlights the relevance of establishing a genetic relationship between a surrogate mother and the child she delivered, given the increasingly strict regulation of surrogacy.

The absence of a common mixed blood flow in the placenta and its separation into fetal and maternal components allows obtaining individual genetic profiles that unequivocally prove a woman’s biological relationship with the child she gave birth to [5].

This work presents three cases that demonstrate challenges in expert evaluation when using different biological materials as reference samples.

Case Description

The article discusses three cases of forensic molecular genetic examination conducted in 2023–2024, which were initiated by Russia’s Investigative Committee.

Several years ago, IVF procedures involving foreign citizens as sperm donors were performed in St. Petersburg. The children were subsequently adopted by their biological parents and relocated to other countries. The investigators argued that the newborns were biological children of women who carried them (i.e., surrogate mothers), which may constitute a violation of the existing Russian legislation. As a result, criminal cases were launched, and genetic examinations were performed to establish the relationship between surrogate mothers and the children they delivered.

Forensic Examinations

In Russia, blood samples are collected from newborns to test for genetic abnormalities. Furthermore, placenta and umbilical cord samples are taken for histopathological examinations to detect intrauterine abnormalities⁶. These biological samples are retained for some time and can be used for molecular genetic testing.

• The following was provided for the forensic examinations in question:
• Three samples of buccal epithelium obtained from surrogate mothers K., E., and D. during the preliminary investigation;
• Three blood samples from newborns, which were dried on special filter cards;

Paraffin blocks with placenta samples from surrogate mothers K., E., and D. obtained after delivery.

To enable an objective examination with clear conclusions, the histopathological analysis involved establishing the position of maternal and fetal fragments in paraffin blocks with placenta samples and labeling them.

During the histopathological examination, the provided paraffin blocks were used to prepare serial sections on a Leica SM2000R^® sliding microtome (Leica Biosystems, Germany). The sections were stained with hematoxylin and eosin using a Leica ST5010^® autostainer (Leica Biosystems, Germany). During microscopy, the maternal and fetal portions of the placenta were labeled.

During molecular genetic testing, DNA was isolated from paraffin blocks using the Thermo Scientific^® GeneJET FFPE DNA Purification Kit (Thermo Fisher Scientific Baltics UAB, Lithuania), as well as from buccal epithelium of surrogate mothers K., E., and D. and blood samples of the children using 5% Chelex-100 suspension (Bio-Rad, USA).

The template activity of DNA samples was assessed by polymerase chain reaction using the Quantifiler TRIO DNA Quantification Kit^® (Applied Biosystems, USA) on a QuantStudio^® 5 System amplifier (Applied Biosystems, USA) with the HID Real-Time PCR Analysis Software v1.3 (Applied Biosystems, USA).

Chromosomal DNA genotyping was performed using the COrDIS Expert 26^® reagent kit for a multiplex assay of 26 human DNA markers (Gordiz, Russia). The kit includes 22 pairs of autosomal loci, sex-specific loci SRY, Yindel, and DYS391, and a segment of the amelogenin gene on chromosomes X and Y. GeneMapper^® ID Software v3.2 and ID-X v1.6 (Applied Biosystems, USA) were used for the analysis.

Molecular Genetic Testing Findings

Total cellular DNA was isolated from biological samples (histological blocks) of the maternal and fetal portions of the placenta from surrogate mothers K., E., and D. according to the labeling of histological sections (Fig. 1), as well as from buccal epithelium samples of surrogate mothers K., E., and D. and blood samples of the children. The DNA was examined using identifying molecular genetic systems based on chromosomal DNA amplified fragment length polymorphism (AFLP). Furthermore, a comparative assessment of these samples was performed.

 

Fig. 1. Location of marked objects: a — on a paraffin block with placental tissues; b — on a histological preparation (stained with hematoxylin and eosin): 1 — fetal part of the placenta; 2 — maternal part of the placenta.

 

Male and female sex was reported for two and one genetic profile of the fetal portion of the placenta, respectively. Alleles absent in the genotypes of surrogate mothers K., E., and D. were identified based on short tandem repeat (STR) loci, indicating that they originated from other women (i.e., biological mothers of the newborns). Therefore, surrogate mothers K., E., and D. were ruled out as biological mothers of children whose fetal portions of the placenta were examined. The analysis confirmed that other women were the biological mothers of the newborns whose genetic material was identified in fetal portions of the placenta from surrogate mothers K., E., and D.

Each sample of the DNA isolated from maternal portions of the placenta from surrogate mothers K. and E. is a mixture of two individual DNAs (male and female).

Furthermore, the genetic profile of the maternal portion of the placenta from surrogate mother D. was completely identical to the AFLP profile of her buccal epithelium sample.

The analysis of autosomal genetic characteristics revealed that maternal portions of the placenta from surrogate mothers K. and E. may contain the genetic material of these surrogate mothers and a male individual (newborn), whose genetic material is present in the fetal portion of the placenta from surrogate mothers K. and E., respectively.

The genetic profiles of blood samples from children born from surrogate mothers E. and D. were completely identical to the genotypes identified in fetal portions of the respective placenta samples.

The genetic profile of the fetal portion of the placenta from surrogate mother D. contains an individual female DNA. Alleles absent in the genotype of surrogate mother D. were identified based on STR loci, indicating that they originated from other women (i.e., the biological mother of the newborn). Therefore, surrogate mother D. was ruled out as the biological mother of the child she delivered. Furthermore, the genetic profile of the maternal portion of the placenta from surrogate mother D. was completely identical to the AFLP profile of her buccal epithelium sample. In this case, the genetic profile of the provided child’s blood sample is completely identical to the genotype found in the fetal portion of the placenta from surrogate mother D.

The genetic profile of the fetal portion of the placenta from surrogate mother E. contained an individual female DNA. Alleles absent in the genotype of surrogate mother E. were identified based on STR loci, indicating that they originated from other women (i.e., the biological mother of the newborn). Therefore, surrogate mother E. was ruled out as the biological mother of the child she delivered. The DNA sample isolated from the maternal portion of the placenta from surrogate mother E. is a mixture of two individual DNAs (male and female). The genetic testing revealed that this mixture may contain the genetic material of surrogate mother E. and a male individual (newborn), whose genetic material is present in the fetal portion of the placenta from surrogate mother E. In this case, the genetic profile of the provided child’s blood sample is completely identical to the genotype found in the fetal portion of the placenta.

The genetic profiles of blood samples from children born from surrogate mothers E. and D., respectively, were completely identical to the genotypes identified in fetal portions of the respective placenta samples.

The allele combinations identified in DNA samples isolated from paraffin blocks of placenta tissues from surrogate mothers D. and E., as well as from buccal epithelium samples of surrogate mothers D. and E. and blood samples of the children, are presented in Tables 1 and 2, respectively.

 

Table 1. Genotypic allele combinations of DNA profiles based on amplified fragment length polymorphism identified during biological material examinations (surrogate mother D.)

Locus	Objects
	Buccal epithelium sample, surrogate mother D.	Maternal portion of the placenta	Fetal portion of  the placenta	Child’s blood  sample
AMEL	ХХ	ХХ	ХХ	ХХ
SRY	—	—	—	—
D3S1358	14.17	14.17	17.18	17.18
TH01	9.9.3	9.9.3	8.9	8.9
D12S391	17.18	17.18	18.20	18.20
D5S818	12.13	12.13	10.11	10.11
TPOX	10.11	10.11	8.9	8.9
Yindel	—	—	—	—
D2S441	11.14	11.14	11.11	11.11
D7S820	9.10	9.10	8.11	8.11
D13S317	11.13	11.13	8.11	8.11
FGA	23.24	23.24	19.23	19.23
D22S1045	12.15	12.15	15.16	15.16
D18S51	15.18	15.18	17.20	17.20
D16S539	11.12	11.12	9.13	9.13
D8S1179	12.16	12.16	11.13	11.13
CSF1PO	10.11	10.11	11.12	11.12
D6S1043	11.12	11.12	11.12	11.12
vWA	14.16	14.16	16.17	16.17
D21S11	27.28	27.28	29.29	29.29
SE33	16.23.2	16.23.2	19.23.2	19.23.2
D10S1248	13.16	13.16	13.14	13.14
D1S1656	15.3.16.3	15.3.16.3	13.15.3	13.15.3
D19S433	16.16	16.16	13.14	13.14
D2S1338	17.20	17.20	17.18	17.18
DYS391	—	—	—	—

 

Table 2. Genotypic allele combinations of DNA profiles based on amplified fragment length polymorphism identified during biological material examinations (surrogate mother E.)

Locus	Objects
	Buccal epithelium sample, surrogate mother D.	Maternal portion of the placenta	Fetal portion of the placenta	Child’s blood sample
AMEL	ХХ	ХY	ХY	ХY
SRY	—	+	+	+
D3S1358	15.17	15.16.17.18	16.18	16.18
TH01	6.9.3	6.7.9.9.3	7.9	7.9
D12S391	18.19	18.19.20	20.20	20.20
D5S818	11.11	11.12	11.12	11.12
TPOX	8.11	8.9.11	8.*	8.9
Yindel	—	1	1	1
D2S441	11.11.3	11.11.3	11.11	11.11
D7S820	9.10	8.9.10.11	8.11	8.11
D13S317	11.13	8.11.13	8.11	8.11
FGA	22.25	19.22.25	19.23	19.23
D22S1045	15.16	15.16	15.16	15.16
D18S51	14.18	14.15.18	14.15	14.15
D16S539	9.11	9.11.12	9.12	9.12
D8S1179	13.15	13.15	13.15	13.15
CSF1PO	10.11	10.11	11.12	11.12
D6S1043	12.20	20.*	12.*	12.20
vWA	14.16	14.16.17	16.17	16.17
D21S11	30.30.2	29.30.30.2.32.2	29.32.2	29.32.2
SE33	17.34.2	14.17.27.2.34.2	14.27.2	14.27.2
D10S1248	12.13	12.13.14.15	14.15	14.15
D1S1656	13.17.3	13.17.3.18.3	13.18.3	13.18.3
D19S433	13.16	13.14.16	13.14	13.14
D2S1338	13.18	13.18	17.18	17.18
DYS391	—	—	11	11

Note. *, any allele of this locus.

 

The genetic profile of the fetal portion of the placenta from surrogate mother K. contains an individual male DNA. Alleles absent in the genotype of surrogate mother K. were identified based on STR loci, indicating that they originated from other women (i.e., the biological mother of the newborn). Therefore, surrogate mother K. was ruled out as the biological mother of the child she delivered. The DNA sample isolated from the maternal portion of the placenta from surrogate mother K. is a mixture of two individual DNAs (male and female). The genetic testing revealed that this mixture may contain the genetic material of surrogate mother K. and a male individual (newborn), whose genetic material is present in the fetal portion of the placenta from surrogate mother K.

However, the DNA isolated from the blood sample of the child presumably born from surrogate mother K. was female, which contradicts the examination concept. A comparison between the genotype identified in the provided child’s blood sample and the AFLP DNK profile of the fetal portion of the placenta from surrogate mother K. showed mismatching genetic traits. Therefore, the genetic material in the placenta from surrogate mother K. and the provided child’s blood sample is from different persons. Thus, the blood sample does not belong to the child delivered by surrogate mother K.

The allele combinations identified in DNA samples isolated from paraffin blocks of placenta tissues from surrogate mother K., as well as from buccal epithelium samples of surrogate mother K. and blood samples of the child, are presented in Table 3.

 

Table 3. Genotypic allele combinations of DNA profiles based on amplified fragment length polymorphism identified during biological material examinations (surrogate mother K.)

Locus	Objects
	Buccal epithelium sample, surrogate mother D.	Maternal portion of the placenta	Fetal portion of the placenta	Child’s blood sample
AMEL	ХХ	ХY	ХY	ХХ
SRY	—	+	+	—
D3S1358	15.17	15.17	15.17	15.15
TH01	9.3.9.3	7.9.9.3	7.9	6.7
D12S391	18.22	18.22.23	18.23	19.23
D5S818	12.13	12.13	12.13	11.12
TPOX	8.8	8.8	8.11	8.8
Yindel	—	2	2	—
D2S441	13.14	13.14	14.14	10.13
D7S820	9.10	9.10.11	10.11	9.10
D13S317	9.9	9.12	12.12	11.12
FGA	19.21	18.19.21.24	18.24	21.24
D22S1045	11.16	11.16	11.16	15.16
D18S51	15.16	15.16.17	16.17	14.15
D16S539	12.13	8.10.12.13	8.10	12.12
D8S1179	13.14	9.13.14	9.13	13.13
CSF1PO	10.10	10.11.12	12.12	9.11
D6S1043	11.17	11.13.17	11.13	12.19
vWA	18.20	16.18.19.20	16.19	17.17
D21S11	29.31.2	28.29.31.2	28.31.2	28.30.2
SE33	27.2.29.2	22.2.27.27.2.29.2	22.2.27	15.21
D10S1248	14.15	14.15.16	14.16	13.15
D1S1656	12.17	11.12.17	11.12	12.17.3
D19S433	13.15	13.14.14.2.15	14.14.2	13.14
D2S1338	25.25	23.25	23.23	18.24
DYS391	—	10	10	—

 

DISCUSSION

This molecular genetic examination of placenta tissues can confirm or rule out the biological relationship in order to meet the legal requirements for surrogacy. The fetal and maternal portions of the placenta contain the material of genetically unrelated individuals (in gestational surrogacy). However, placenta samples must be used in molecular genetic testing to confirm that a surrogate mother delivered a particular child. Alleles that differ from the surrogate mother’s genetic traits were identified in the fetal and maternal portions of the placenta, indicating that they may have originated from the biological mother. The use of placenta tissues as samples of the newborn’s biological materials confirms that the surrogate mother is not biologically related to the child. This child was born from a particular surrogate mother; however, despite the genetic differences, they are not biologically related.

Molecular genetic testing of placenta samples allows confirming the source of the paraffin-embedded biological material, enabling the expert to confirm that the child was born from this particular woman.

Furthermore, a comparison with a sample from a person who is not directly related to the surrogate mother will rule her out as the biological mother. This could lead the investigators to erroneous conclusions.

The presented case highlights the importance of ruling out errors (including human errors, deliberate substitution of a sample, etc.) when establishing the genetic relationship using placenta tissues as reference samples. Notably, such cases have not previously been addressed in scientific publications.

CONCLUSION

All three cases rule out surrogate mothers as biological mothers of the children they delivered, which meets the requirement of Russian legislation. However, one of these cases demonstrates mismatching DNA profiles of the provided reference sample of the newborn and the true genotype of the child delivered by this particular surrogate mother.

Therefore, paraffin-embedded placenta samples can be used to rule out errors in establishing maternity in nonobvious cases.

The examinations allowed the investigators to determine the true origin of the children relocated from Russia by biological parents who adopted them, as well as rule out any violations of legislation by surrogate mothers.

ADDITIONAL INFORMATION

Author contributions: A.V. Konovalenko: conceptualization, investigation, writing—review & editing; I.I. Kukharenok: investigation, writing—original draft, writing—review & editing. All the authors approved the version of the manuscript to be published and agreed to be accountable for all aspects of the work, ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

Acknowledgments: The authors express their special gratitude to Professor P.L. Ivanov for his invaluable support and guidance during the manuscript preparation, as well as to A.F. Titievskaya, Head of the Forensic Histology Department of the Bureau of Forensic Medical Examination (St. Petersburg), for her cooperation with this work.

Ethics approval: Not applicable.

Consent for publication: Written informed consent was not obtained from the individuals or their legal representatives for the publication of expert case materials, as it was not possible to establish contact with them. All data presented are anonymized, and no photographs are published.

Funding sources: No funding.

Disclosure of interests: The authors have no relationships, activities, or interests for the last three years related to for-profit or not-for-profit third parties whose interests may be affected by the content of the article.

Statement of originality: No previously published materials (text, figures, or data) were used in this work.

Data availability statement: The editorial policy regarding data sharing does not apply to this work.

Generative AI: No generative artificial intelligence technologies were used to prepare this article.

Provenance and peer-review: This article was submitted unsolicited and reviewed following the standard procedure. The peer review process involved two external reviewers and the in-house scientific editor.

 

¹ Preliminary Document No. 10 of March 2012 for the Attention of the Council of April 2012 on General Affairs and Policy of the Conference [Internet]. Hague: Permanent Bureau of the Hague Conference on Private International Law, 2012–2024. Available at: https://assets.hcch.net/docs/. Accessed on: December 10, 2024.

² Surrogacy in Russia [Internet]. In: Wikipedia. 2022–. Available at: https://ru.wikipedia.org/wiki/. Accessed on: December 10, 2024.

³ Order of the Ministry of Health of the Russian Federation No. 107n of August 30, 2012, On the Use of Assisted Reproductive Technology, Contraindications, and Limitations (annulled). Available at: https://base.garant.ru/70318364/. Accessed on: December 10, 2024.

⁴ Federal Law of the Russian Federation No. 323-FZ of November 21, 2011, On Fundamental Healthcare Principles in the Russian Federation. Available at: http://www.rosminzdrav.ru/documents/7025-federalnyy-zakon-323-fz-ot-21-noyabrya-2011-g. Accessed on: December 10, 2024.

⁵ Order of the Ministry of Health of the Russian Federation No. 803n, On the Use of Assisted Reproductive Technology, Contraindications, and Limitations. Available at: https://www.garant.ru/products/ipo/prime/doc/74676088/. Accessed on: December 10, 2024.

⁶ Order of the Ministry of Health of the Russian Federation No. 274n of April 21, 2022, On Approval of the Procedure for Medical Care in Patients With Congenital and/or Hereditary Disorders. Available at: https://base.garant.ru/404987183/. Accessed on: December 10, 2024.

About the authors

Andrey V. Konovalenko

Bureau of Forensic Medical Examination; Saint-Petersburg State Pediatric Medical University

Author for correspondence.
Email: andrkon1980@mail.ru
ORCID iD: 0009-0002-0306-423X

MD

Russian Federation, Saint Petersburg; Saint Petersburg

Irina I. Kukharenok

Bureau of Forensic Medical Examination

Email: irishka2402@mail.ru
ORCID iD: 0009-0007-2645-0025
SPIN-code: 2501-0929

MD

Russian Federation, Saint Petersburg

References

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