A case report of twin-chorionic triamniotic triplet pregnancy following single blastocyst transfer in a thin endometrium

A case report of twin-chorionic triamniotic triplet pregnancy following single blastocyst transfer in a thin endometrium | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Case Report A case report of twin-chorionic triamniotic triplet pregnancy following single blastocyst transfer in a thin endometrium Yuxing Xiong, Mei Tang, Sha Shi, Yan Liu This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8428992/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 8 You are reading this latest preprint version Abstract Background Endometrial thickness is a key index to evaluate endometrial receptivity, which is closely related to embryo implantation and pregnancy outcome. Thin or damaged endometrium can lead to embryo implantation failure and early pregnancy abortion. It is extremely rare for patients with thin endometrium to have multiple pregnancies. Case presentation A 35-year-old woman with primary infertility for 1 year, thin endometrium caused by chronic endometritis, and combined with a variety of other infertility factors. In the fresh cycle, 8 oocytes were obtained by short-acting long protocol, and 7 embryos were frozen. The fresh cycle transplantation was cancelled due to insufficient endometrial thickness (6.7 mm). The endometrial microenvironment was improved by comprehensive treatment during the resuscitation cycle. After the endometrium was increased to 8.3 mm, the single blastocyst was resuscitated and transplanted, and the triplet pregnancy of double chorionic triple amniotic sac was obtained. After natural reduction, a healthy baby boy was delivered at term. Conclusions Even if single blastocyst transplantation, there is still an unpredictable risk of single oocyte division, which can lead to high-order multiple pregnancy. Clinically, patients should be fully informed of the risk, and closely monitored during early pregnancy. If necessary, selective reduction should be performed in time to improve maternal and infant outcomes. Thin endometrium Multiple pregnancy Frozen-thawed embryo transfer Natural reduction of tire Figures Figure 1 Introduction The incidence of thin endometrium (TE) in patients undergoing assisted reproductive technology is 2.4% -8.5%. At present, there is no unified definition of thin endometrium. It is believed that thin endometrium refers to the determination of endometrial thickness ≤ 7mm on the day of oocyte retrieval, the day of trigger of fresh embryo transfer cycle or the day of endometrial transformation in frozen-thawed embryo transfer (FET) cycle [ 1 ]. The clinical pregnancy rate and live birth rate are positively correlated with endometrial thickness, but it is necessary to comprehensively evaluate the morphology, type and blood flow of the endometrium [ 2 ]. This paper reports a case of thin endometrium patient who developed into a double chorionic triple amniotic sac embryo after one blastocyst through in vitro fertilization-embryo transfer (IVF-ET), which adds a new case to the rare monozygotic multiple pregnancy in assisted reproductive technology (ART), in order to provide reference for clinical diagnosis and treatment. Case presentation A 35-year-old female patient was admitted to the reproductive center of our hospital due to non-contraception and non-pregnancy for 1 year. The patient had regular menstruation, 14-year-old menarche, 27 days of menstrual cycle, 5 days of menstrual period, moderate menstrual volume, and no dysmenorrhea. Last menstruation : June 22,2024. In May 2024, the basic sex hormone examination was performed : follicle-stimulating hormone (FSH) : 5.76 mIU / ml, luteinizing hormone (LH) :6.18 mIU / ml, estradiol (E2) :56.85 pg / ml, progesterone (P) :0.18ng / ml, testosterone (T) :0.27ng / ml, prolactin (PRL) :19.58ng / ml, anti-millerian hormone (AMH) :1.010ng / ml. The number of antral follicles was 4 on the left and 5 on the right. In our hospital, multi-cycle drug ovulation induction was used to monitor follicular development, and all of them had dominant follicular ovulation to guide non-pregnancy in the same room. In June 2024, hysterosalpingography was performed in our hospital. The results showed that the uterus was normal, the left fallopian tube was unobstructed, and the proximal end of the right fallopian tube was completely obstructed.In July 2024, hysteroscopy and endometrial biopsy were performed for multiple endometrial polyps. Immunohistochemical pathological results showed a small amount of plasma cell infiltration, CD38 (a small amount of +), CD138 (-), considered chronic endometritis. In August 2024, the body mass index was 19.77 kg / m2, and there was no abnormality in gynecological examination. Female chromosome : 46, XX, 9qh +. Thyroid function, liver and kidney function, blood routine, TORCH, reproductive immune antibody and cervical liquid-based thin-layer cytology were normal. Male semen examination was normal, chromosome : 46, XY. The patient underwent IVF-ET in the reproductive center of our hospital in September 2024 due to primary infertility and right fallopian tube obstruction. Before IVF-ET, chronic endometritis was treated with doxycycline and metronidazole for anti-inflammatory treatment. During the medication period, strict contraception was performed, and oral short-acting contraceptives were taken for one cycle to repair the endometrium. The patient was short-acting long-acting protocol. Controlled ovarian stimulation was performed with recombinant FSH (Lishenbao) combined with highly purified hMG (Hemeiqi), followed by triggering with 6,500 IU recombinant HCG (Lidebao). Oocyte retrieval was carried out 36.5 hours later.A total of 8 oocytes were retrieved vaginally under ultrasound guidance, of which 8 oocytes matured and 8oocytes were successfully fertilized and cleavaged. In vitro, embryo culture showed two high-quality embryos on day 3.All embryos were cultured in vitro to day 5 , resulting in a total of 5 blastocysts.The endometrium was primed with intrauterine perfusion of somatropin (Ansumeng) 6 IU daily for 8 days; maximum thickness achieved was 6.7 mm, which was judged out of phase with follicular maturation. In view of the well-documented negative impact of a thin endometrium on implantation and clinical pregnancy rates, and after thorough counselling and informed consent, fresh embryo transfer was cancelled and all viable embryos were cryopreserved. Frozen–thawed embryo transfer cycle was initiated in November 2024. Baseline endometrial thickness was 4 mm. Estradiol valerate/dydrogesterone (Femoston) was prescribed to promote endometrial proliferation; low-dose acetylsalicylic acid (Bayer Aspirin) was added for thromboprophylaxis and to enhance endometrial perfusion, thereby potentiating the estrogenic response. Three intrauterine perfusions of somatropin were administered to optimize the endometrial microenvironment, and sildenafil (Viagra) 25 mg was given vaginally to further improve uterine blood flow. Serial monitoring demonstrated endometrial thickness increased to 8.3 mm, with serum LH:0.56 IU/L, E₂:1,111 pg/mL, and P:0.24 ng/mL. On November 11, 2024, one blastocyst (4AA) was transplanted and luteal support was given after operation. Serum HCG 14 days after transfer was 479.1 IU/L.On the 31 st day after transplantation, vaginal B-ultrasound examination showed that two gestational sacs were detected in the uterine cavity, with a size of about 41 mm × 22 mm (GS1) and 15 mm × 12 mm (GS2), respectively. Two yolk sacs, germ tissue and original heart tube pulsation were observed in GS1, and the length of germs was 76 mm and 44 mm, respectively. Yolk sac and germ tissue and original heart tube pulsation were found in GS2, and the length of germ was 41 mm(Fig.1A).On the 42 nd day after transplantation, vaginal B-ultrasound examination showed that 45mm × 32mm (GS1) and 14mm × 9mm (GS2) gestational sacs were found in the uterine cavity, with no echo, yolk sac and embryos with length of 21mm and 7mm, respectively. The former showed cardiac tube pulsation, while the latter showed no obvious cardiac tube pulsation (Fig.1B). B-ultrasound reexamination at 61 days after transplantation showed that the intrauterine single fetus survived and the fetus was naturally reduced (Fig.1C).The follow-up results of regular prenatal examination were normal, and the process of late pregnancy was stable. One male baby was successfully delivered by cesarean section at 38 +5 weeks of pregnancy, with Apgart score of 9 points. The follow-up of children also confirmed that they were in good health. Discussion and conclusions In the process of assisted pregnancy, this case has faced two key problems : thin endometrium and multiple pregnancy after single blastocyst transfer. The formation of thin endometrium may be related to many factors, and its etiology can be classified into three categories : inflammatory, iatrogenic and idiopathic [1].Although the etiology is heterogeneous, its clinical outcomes are similar, embryo implantation rate is reduced, and the risk of abortion is increased [3,4]. The patient had a history of intrauterine operation and chronic endometritis, which may damage the blood vessels and glands in the basal layer of the intima, resulting in a decrease in the reactivity of the intima to estrogen, which is an important cause of poor intima proliferation in the fresh cycle.This history indicates that endometrial factors should be the primary focus during the frozen–thawed replacement cycle.A multi-modal regimen was therefore instituted: estrogen to drive proliferation, low-dose aspirin to improve perfusion and thromboprophylaxis while potentiating the endometrial response to estradiol, and vaginal sildenafil to dilate uterine arteries and further increase blood flow.On this basis, intrauterine perfusion of growth hormone improves the endometrial microenvironment. Intrauterine perfusion can achieve direct action of drugs on the endometrium, thus giving full play to its biological effects [5]. After a series of treatments, the endometrial thickness of the patients increased from 6.7 mm to 8.3 mm, and the single blastocyst resuscitation transplantation was successfully completed.For TE caused by chronic endometritis, combined with growth hormone intrauterine perfusion on the basis of anti-inflammatory can significantly improve endometrial reactivity, which is worthy of verification in larger samples in the future. Multiple pregnancy is defined as pregnancy with two or more fetuses, such as triplets or quadruplets [6]. With the development of assisted reproductive technology and the wide application of ovulation-promoting drugs, the incidence of multiple pregnancies has increased [7]. The specific reason is unknown, and it is preliminarily inferred that it is related to maternal age, blastocyst culture, ovulation induction therapy, assisted hatching, frequent zona pellucida operation and medium conditions [8].The division of inner cell mass and blastocyst culture during in vitro culture are most often associated with multiple pregnancies. Prolonged culture to the blastocyst stage may lead to zona pellucida hardening, so that the inner cell mass is squeezed or divided during embryo hatching [9]. In order to reduce the incidence of multiple pregnancies, single blastocyst transfer has become a routine method, but it cannot completely avoid the occurrence of multiple pregnancies [10].In this case, the rare double chorion three amniotic sac pregnancy occurred in the context of single blastocyst transfer. The patient was 35 years old. IVF fertilization could rule out the influence of intracytoplasmic sperm injection(ICSI) operation on the zona pellucida structure. It was believed that the assisted hatching of the resuscitation cycle may be the cause of monozygotic multiple births in this case. Multiple pregnancy is a high-risk pregnancy, which is prone to serious complications such as premature delivery, abortion and gestational hypertension.Multifetal reduction is a common clinical intervention, but the effect of different timing of reduction on pregnancy outcome is controversial. Studies have shown that in order to achieve better pregnancy outcomes, fetal reduction can be performed as soon as possible, so that the uterus is less stimulated, the operation is easier, and the residual necrotic tissue of the reduced fetus is less [11]. Anthoulakis C et al.showed that early fetal reduction would increase the risk of abortion [12]. Fortunately, the patient in this paper naturally reduced the fetus to a single live fetus, avoiding the risk of infection, bleeding and residual fetal loss caused by artificial reduction [13]. The patient also had chromosomal abnormalities. 46, XX, 9qh + refers to the increased length of the heterochromatin region on the long arm of chromosome 9 in the female chromosome karyotype. In a study of 2080 infertile patients, a total of 88 patients with chromosome qh + were detected [14]. In another study, it was found that the incidence of 9qh + in infertile women was 16.22 %. These data indicate that there may be a certain correlation between 9qh + and infertility [15]. 9qh + may lead to fertility problems by affecting the meiosis process. The increase of heterochromosome regions may lead to abnormal pairing of homologous chromosomes, forming a special inverted ring structure, affecting the expression of nearby functional genes and thus affecting the development and function of germ cells. The patient carried 9qh + abnormal chromosome, suggesting that it may be related to infertility. In summary, it is necessary to comprehensively evaluate the physical condition and personal willingness of different patients, and formulate individualized treatment plans based on the etiology and treatment effect. Improving ART pregnancy rate, obtaining single embryo pregnancy and reducing complications are the goals pursued in the field of assisted reproduction. TE patients should be fully informed of the treatment difficulties, the advantages and disadvantages of treatment options, and possible prognosis. Growth hormone intrauterine perfusion therapy shows a good application prospect. Granulocyte colony-stimulating factor, autologous platelet-rich and stem cell therapy also provide a new direction for endometrial repair. It is worth noting that even if the number of embryos transferred is limited, the risk of multiple pregnancies cannot be completely avoided, and the possibility of spontaneous embryo division still exists. Risk should be fully informed before treatment. Blood β-hCG and ultrasound should be closely monitored after transplantation. Pregnancy and embryonic development status should be identified as early as possible, and individualized management should be implemented to optimize pregnancy outcomes. Future studies should further verify the potential effects of various intervention factors on embryo division, and provide a more solid theoretical basis for improving the effectiveness and safety of ART. Abbreviations TE The incidence of thin endometrium FET Frozen-thawed embryo transfer IVF-ET In vitro fertilization-embryo transfer ART Assisted reproductive technology ICSI Intracytoplasmic sperm injection FSH Follicle-stimulating hormone LH Luteinizing hormone E 2 Estradiol P Progesterone T Testosterone PRL Prolactin AMH Anti-millerian hormone HCG Human chorionic gonadotropin GS Gestational sacs Declarations Acknowledgements We would like to thank the patient and her family for consenting to publish this case report. Author contributions Y. X.was responsible for writing the first draft and the major revisions of the report;Y.L. was mainly responsible for supervising the research, conception, and review of papers; M.T. and S.S were responsible for literature collection and collation. All authors read and approved the final version of the manuscript. Funding This research was funded by the Health and Hygiene Research Program,Metallurgical Safety and Health Branch,Chinese Society for Metals,grant Number jkws202351,the Wuhan Medical Science Research Program,grant Number WX23Z24. Availability of data and materials All data was included in the published manuscript. Consent for publication Written informed consent for publication was obtained from the patient. Competing interests The authors declare no competing interests. Ethics approval and consent to participate This study was approved by the Ethics Committee of Puren Hospital Affiliated to Wuhan University of Science and Technology(PR20240908). Written consent was obtained from all the parents. References Liu KE, Hartman M, Hartman A. Management of thin endometrium in assisted reproduction: a clinical practice guideline from the Canadian Fertility and Andrology Society. Reprod Biomed Online. 2019;39(1):49–62. Salman MM, Zaki AM, El-Gamal HH, et al. Effect of intrauterine infusion of autologous platelet-rich plasma in patients with refractory thin endometrium undergoing in vitro fertilization. Prz Menopauzalny. 2023;22(2):77–82. Liao Z, Liu C, Cai L, et al. The Effect of Endometrial Thickness on Pregnancy, Maternal, and Perinatal Outcomes of Women in Fresh Cycles After IVF/ICSI: A Systematic Review and Meta-Analysis. Front Endocrinol (Lausanne). 2022;12:814648. Pirtea P, Cicinelli E, De Nola R, et al. Endometrial causes of recurrent pregnancy losses: endometriosis, adenomyosis, and chronic endometritis. Fertil Steril. 2021;115(3):546–60. Li W, Cao Z, Yu X, et al. Effect of growth hormone on thin endometrium via intrauterine infusion. Ann Transl Med. 2021;9(16):1325. Seetho S, Kongwattanakul K, Saksiriwuttho P, et al. Epidemiology and factors associated with preterm births in multiple pregnancy: a retrospective cohort study. BMC Pregnancy Childbirth. 2023;23(1):872. Sunderam S, Kissin DM, Zhang Y, et al. Assisted Reproductive Technology Surveillance - United States, 2018. MMWR Surveill Summ. 2022;71(4):1–19. Jin H, Han Y, Zenker J. Cellular mechanisms of monozygotic twinning: clues from assisted reproduction. Hum Reprod Update. 2024;30(6):692–705. Nakasuji T, Saito H, Araki R, et al. The incidence of monozygotic twinning in assisted reproductive technology: analysis based on results from the 2010 Japanese ART national registry. J Assist Reprod Genet. 2014;31(7):803–7. Scaravelli G, Pisaturo V, Levi Setti PE, et al. Monozygotic twin rate among ART centers: a multicenter analysis of data from 18 Italian units. J Assist Reprod Genet. 2022;39(10):2349–54. Zou G, Ji Q, Chen J, et al. Perinatal outcome and timing of selective fetal reduction in dichorionic diamniotic twin pregnancies: a single-center retrospective study. Front Med (Lausanne). 2024;10:1327191. Cai P, Ouyang Y, Gong F, et al. Pregnancy outcomes of dichorionic triamniotic triplet pregnancies after in vitro fertilization-embryo transfer: multifoetal pregnancy reduction versus expectant management. BMC Pregnancy Childbirth. 2020;20(1):165. Meireson E, De Rycke L, Bijnens EM, et al. Birth outcomes of twins after multifetal pregnancy reduction compared with primary twins. Am J Obstet Gynecol MFM. 2024;6(1):101230. Lu Y, Tian T, Chen L, et al. Diverse impacts of female chromosomal polymorphisms on assisted reproduction outcomes: a retrospective cohort study. BMC Pregnancy Childbirth. 2024;24(1):331. Xie X, Li F, Tan W, et al. Analysis of the clinical features of pericentric inversion of chromosome 9. J Int Med Res. 2020;48(9):300060520957820. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Reviews received at journal 28 Jan, 2026 Reviewers agreed at journal 25 Jan, 2026 Reviewers agreed at journal 21 Jan, 2026 Reviewers invited by journal 20 Jan, 2026 Editor assigned by journal 19 Jan, 2026 Editor invited by journal 29 Dec, 2025 Submission checks completed at journal 24 Dec, 2025 First submitted to journal 24 Dec, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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00:39:15","extension":"html","order_by":15,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":46135,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8428992/v1/5fb971bbac9dc301bdfd1910.html"},{"id":100930096,"identity":"193f26d8-5518-4113-8dbb-fdb0b758393a","added_by":"auto","created_at":"2026-01-23 00:39:30","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":550069,"visible":true,"origin":"","legend":"\u003cp\u003eA) Vaginal ultrasound reveals dichorionic triamniotic triplets with two gestational sacs (GS1,GS2) and three germsin the uterus. B)Ultrasound image was taken for 42 days after transplantation. C)Ultrasound image was taken for day 61 after transplantation.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8428992/v1/37b7dcdb560597fe77bbafd6.png"},{"id":100930166,"identity":"6f46c475-882f-4155-8ece-b069bbaa14bf","added_by":"auto","created_at":"2026-01-23 00:39:54","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1200777,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8428992/v1/ed33a379-19ac-41a3-9991-600158b9cdc2.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"A case report of twin-chorionic triamniotic triplet pregnancy following single blastocyst transfer in a thin endometrium","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe incidence of thin endometrium (TE) in patients undergoing assisted reproductive technology is 2.4% -8.5%. At present, there is no unified definition of thin endometrium. It is believed that thin endometrium refers to the determination of endometrial thickness\u0026thinsp;\u0026le;\u0026thinsp;7mm on the day of oocyte retrieval, the day of trigger of fresh embryo transfer cycle or the day of endometrial transformation in frozen-thawed embryo transfer (FET) cycle [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The clinical pregnancy rate and live birth rate are positively correlated with endometrial thickness, but it is necessary to comprehensively evaluate the morphology, type and blood flow of the endometrium [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. This paper reports a case of thin endometrium patient who developed into a double chorionic triple amniotic sac embryo after one blastocyst through in vitro fertilization-embryo transfer (IVF-ET), which adds a new case to the rare monozygotic multiple pregnancy in assisted reproductive technology (ART), in order to provide reference for clinical diagnosis and treatment.\u003c/p\u003e"},{"header":"Case presentation","content":"\u003cp\u003eA 35-year-old female patient was admitted to the reproductive center of our hospital due to non-contraception and non-pregnancy for 1 year. The patient had regular menstruation, 14-year-old menarche, 27 days of menstrual cycle, 5 days of menstrual period, moderate menstrual volume, and no dysmenorrhea. Last menstruation : June 22,2024. In May 2024, the basic sex hormone examination was performed : follicle-stimulating hormone (FSH) : 5.76 mIU / ml, luteinizing hormone (LH) :6.18 mIU / ml, estradiol (E2) :56.85 pg / ml, progesterone (P) :0.18ng / ml, testosterone (T) :0.27ng / ml, prolactin (PRL) :19.58ng / ml, anti-millerian hormone (AMH) :1.010ng / ml. The number of antral follicles was 4 on the left and 5 on the right. In our hospital, multi-cycle drug ovulation induction was used to monitor follicular development, and all of them had dominant follicular ovulation to guide non-pregnancy in the same room. In June 2024, hysterosalpingography was performed in our hospital. The results showed that the uterus was normal, the left fallopian tube was unobstructed, and the proximal end of the right fallopian tube was completely obstructed.In July 2024, hysteroscopy and endometrial biopsy were performed for multiple endometrial polyps. Immunohistochemical pathological results showed a small amount of plasma cell infiltration, CD38 (a small amount of +), CD138 (-), considered chronic endometritis. In August 2024, the body mass index was 19.77 kg / m2, and there was no abnormality in gynecological examination. Female chromosome : 46, XX, 9qh +. Thyroid function, liver and kidney function, blood routine, TORCH, reproductive immune antibody and cervical liquid-based thin-layer cytology were normal. Male semen examination was normal, chromosome : 46, XY.\u003c/p\u003e\n\u003cp\u003eThe patient underwent IVF-ET in the reproductive center of our hospital in September 2024 due to primary infertility and right fallopian tube obstruction. Before IVF-ET, chronic endometritis was treated with doxycycline and metronidazole for anti-inflammatory treatment. During the medication period, strict contraception was performed, and oral short-acting contraceptives were taken for one cycle to repair the endometrium.\u003c/p\u003e\n\u003cp\u003eThe patient was short-acting long-acting protocol. Controlled ovarian stimulation was performed with recombinant FSH (Lishenbao) combined with highly purified hMG (Hemeiqi), followed by triggering with 6,500 IU recombinant HCG (Lidebao). Oocyte retrieval was carried out 36.5 hours later.A total of 8 oocytes were retrieved vaginally under ultrasound guidance, of which 8 oocytes matured and 8oocytes were successfully fertilized and cleavaged. In vitro, embryo culture showed two high-quality embryos on day 3.All embryos were cultured in vitro to day 5 , resulting in a total of 5 blastocysts.The endometrium was primed with intrauterine perfusion of somatropin (Ansumeng) 6 IU daily for 8 days; maximum thickness achieved was 6.7 mm, which was judged out of phase with follicular maturation. In view of the well-documented negative impact of a thin endometrium on implantation and clinical pregnancy rates, and after thorough counselling and informed consent, fresh embryo transfer was cancelled and all viable embryos were cryopreserved.\u003c/p\u003e\n\u003cp\u003eFrozen\u0026ndash;thawed embryo transfer cycle was initiated in November 2024. Baseline endometrial thickness was 4 mm. Estradiol valerate/dydrogesterone (Femoston) was prescribed to promote endometrial proliferation; low-dose acetylsalicylic acid (Bayer Aspirin) was added for thromboprophylaxis and to enhance endometrial perfusion, thereby potentiating the estrogenic response. Three intrauterine perfusions of somatropin were administered to optimize the endometrial microenvironment, and sildenafil (Viagra) 25 mg was given vaginally to further improve uterine blood flow. Serial monitoring demonstrated endometrial thickness increased to 8.3 mm, with serum LH:0.56 IU/L, E₂:1,111 pg/mL, and P:0.24 ng/mL. On November 11, 2024, one blastocyst (4AA) was transplanted and luteal support was given after operation.\u003c/p\u003e\n\u003cp\u003eSerum HCG 14 days after transfer was 479.1 IU/L.On the 31 st day after transplantation, vaginal B-ultrasound examination showed that two gestational sacs were detected in the uterine cavity, with a size of about 41 mm \u0026times; 22 mm (GS1) and 15 mm \u0026times; 12 mm (GS2), respectively. Two yolk sacs, germ tissue and original heart tube pulsation were observed in GS1, and the length of germs was 76 mm and 44 mm, respectively. Yolk sac and germ tissue and original heart tube pulsation were found in GS2, and the length of germ was 41 mm(Fig.1A).On the 42 nd day after transplantation, vaginal B-ultrasound examination showed that 45mm \u0026times; 32mm (GS1) and 14mm \u0026times; 9mm (GS2) gestational sacs were found in the uterine cavity, with no echo, yolk sac and embryos with length of 21mm and 7mm, respectively. The former showed cardiac tube pulsation, while the latter showed no obvious cardiac tube pulsation (Fig.1B). B-ultrasound reexamination at 61 days after transplantation showed that the intrauterine single fetus survived and the fetus was naturally reduced (Fig.1C).The follow-up results of regular prenatal examination were normal, and the process of late pregnancy was stable. One male baby was successfully delivered by cesarean section at 38\u003csup\u003e+5\u003c/sup\u003e weeks of pregnancy, with Apgart score of 9 points. The follow-up of children also confirmed that they were in good health.\u003c/p\u003e"},{"header":"Discussion and conclusions","content":"\u003cp\u003eIn the process of assisted pregnancy, this case has faced two key problems : thin endometrium and multiple pregnancy after single blastocyst transfer. The formation of thin endometrium may be related to many factors, and its etiology can be classified into three categories : inflammatory, iatrogenic and idiopathic [1].Although the etiology is heterogeneous, its clinical outcomes are similar, embryo implantation rate is reduced, and the risk of abortion is increased [3,4]. The patient had a history of intrauterine operation and chronic endometritis, which may damage the blood vessels and glands in the basal layer of the intima, resulting in a decrease in the reactivity of the intima to estrogen, which is an important cause of poor intima proliferation in the fresh cycle.This history indicates that endometrial factors should be the primary focus during the frozen\u0026ndash;thawed replacement cycle.A multi-modal regimen was therefore instituted: \u0026nbsp;estrogen to drive proliferation, low-dose aspirin to improve perfusion and thromboprophylaxis while potentiating the endometrial response to estradiol, and vaginal sildenafil to dilate uterine arteries and further increase blood flow.On this basis, intrauterine perfusion of growth hormone improves the endometrial microenvironment. Intrauterine perfusion can achieve direct action of drugs on the endometrium, thus giving full play to its biological effects [5]. After a series of treatments, the endometrial thickness of the patients increased from 6.7 mm to 8.3 mm, and the single blastocyst resuscitation transplantation was successfully completed.For TE caused by chronic endometritis, combined with growth hormone intrauterine perfusion on the basis of anti-inflammatory can significantly improve endometrial reactivity, which is worthy of verification in larger samples in the future.\u003c/p\u003e\n\u003cp\u003eMultiple pregnancy is defined as pregnancy with two or more fetuses, such as triplets or quadruplets [6]. With the development of assisted reproductive technology and the wide application of ovulation-promoting drugs, the incidence of multiple pregnancies has increased [7]. The specific reason is unknown, and it is preliminarily inferred that it is related to maternal age, blastocyst culture, ovulation induction therapy, assisted hatching, frequent zona pellucida operation and medium conditions [8].The division of inner cell mass and blastocyst culture during in vitro culture are most often associated with multiple pregnancies. Prolonged culture to the blastocyst stage may lead to zona pellucida hardening, so that the inner cell mass is squeezed or divided during embryo hatching [9]. In order to reduce the incidence of multiple pregnancies, single blastocyst transfer has become a routine method, but it cannot completely avoid the occurrence of multiple pregnancies [10].In this case, the rare double chorion three amniotic sac pregnancy occurred in the context of single blastocyst transfer. The patient was 35 years old. IVF fertilization could rule out the influence of intracytoplasmic sperm injection(ICSI) operation on the zona pellucida structure. It was believed that the assisted hatching of the resuscitation cycle may be the cause of monozygotic multiple births in this case. Multiple pregnancy is a high-risk pregnancy, which is prone to serious complications such as premature delivery, abortion and gestational hypertension.Multifetal reduction is a common clinical intervention, but the effect of different timing of reduction on pregnancy outcome is controversial. Studies have shown that in order to achieve better pregnancy outcomes, fetal reduction can be performed as soon as possible, so that the uterus is less stimulated, the operation is easier, and the residual necrotic tissue of the reduced fetus is less [11]. Anthoulakis C et al.showed that early fetal reduction would increase the risk of abortion [12]. Fortunately, the patient in this paper naturally reduced the fetus to a single live fetus, avoiding the risk of infection, bleeding and residual fetal loss caused by artificial reduction [13].\u003c/p\u003e\n\u003cp\u003eThe patient also had chromosomal abnormalities. 46, XX, 9qh + refers to the increased length of the heterochromatin region on the long arm of chromosome 9 in the female chromosome karyotype. In a study of 2080 infertile patients, a total of 88 patients with chromosome qh + were detected [14]. In another study, it was found that the incidence of 9qh + in infertile women was 16.22 %. These data indicate that there may be a certain correlation between 9qh + and infertility [15]. 9qh + may lead to fertility problems by affecting the meiosis process. The increase of heterochromosome regions may lead to abnormal pairing of homologous chromosomes, forming a special inverted ring structure, affecting the expression of nearby functional genes and thus affecting the development and function of germ cells. The patient carried 9qh + abnormal chromosome, suggesting that it may be related to infertility.\u003c/p\u003e\n\u003cp\u003eIn summary, it is necessary to comprehensively evaluate the physical condition and personal willingness of different patients, and formulate individualized treatment plans based on the etiology and treatment effect. Improving ART pregnancy rate, obtaining single embryo pregnancy and reducing complications are the goals pursued in the field of assisted reproduction. TE patients should be fully informed of the treatment difficulties, the advantages and disadvantages of treatment options, and possible prognosis. Growth hormone intrauterine perfusion therapy shows a good application prospect. Granulocyte colony-stimulating factor, autologous platelet-rich and stem cell therapy also provide a new direction for endometrial repair. It is worth noting that even if the number of embryos transferred is limited, the risk of multiple pregnancies cannot be completely avoided, and the possibility of spontaneous embryo division still exists. Risk should be fully informed before treatment. Blood \u0026beta;-hCG and ultrasound should be closely monitored after transplantation. Pregnancy and embryonic development status should be identified as early as possible, and individualized management should be implemented to optimize pregnancy outcomes. Future studies should further verify the potential effects of various intervention factors on embryo division, and provide a more solid theoretical basis for improving the effectiveness and safety of ART.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eTE \u0026nbsp; \u0026nbsp; \u0026nbsp;The incidence of thin endometrium\u003c/p\u003e\n\u003cp\u003eFET \u0026nbsp; \u0026nbsp; Frozen-thawed embryo transfer\u003c/p\u003e\n\u003cp\u003eIVF-ET \u0026nbsp;In vitro fertilization-embryo transfer\u003c/p\u003e\n\u003cp\u003eART \u0026nbsp; \u0026nbsp; Assisted reproductive technology\u003c/p\u003e\n\u003cp\u003eICSI \u0026nbsp; \u0026nbsp; \u0026nbsp;Intracytoplasmic sperm injection\u003c/p\u003e\n\u003cp\u003eFSH \u0026nbsp; \u0026nbsp; Follicle-stimulating hormone\u003c/p\u003e\n\u003cp\u003eLH \u0026nbsp; \u0026nbsp; \u0026nbsp;Luteinizing hormone\u003c/p\u003e\n\u003cp\u003eE\u003csub\u003e2\u003c/sub\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Estradiol\u003c/p\u003e\n\u003cp\u003eP \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Progesterone\u003c/p\u003e\n\u003cp\u003eT \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Testosterone\u003c/p\u003e\n\u003cp\u003ePRL \u0026nbsp; \u0026nbsp; \u0026nbsp; Prolactin\u003c/p\u003e\n\u003cp\u003eAMH \u0026nbsp; \u0026nbsp; Anti-millerian hormone\u003c/p\u003e\n\u003cp\u003eHCG \u0026nbsp; \u0026nbsp; Human chorionic gonadotropin\u003c/p\u003e\n\u003cp\u003eGS \u0026nbsp; \u0026nbsp; \u0026nbsp; Gestational sacs\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to thank the patient and her family for consenting to publish this case report.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eY. X.was responsible for writing the first draft and the major revisions of the report;Y.L. was mainly responsible for supervising the research, conception, and review of papers; M.T. and S.S were responsible for literature collection and collation. All authors read and approved the final version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was funded by the Health and Hygiene Research Program,Metallurgical Safety and Health Branch,Chinese Society for Metals,grant Number jkws202351,the Wuhan Medical Science Research Program,grant Number WX23Z24.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data was included in the published manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWritten informed consent for publication was obtained from the patient.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was approved by the Ethics Committee of Puren Hospital Affiliated to Wuhan University of Science and Technology(PR20240908). Written consent was obtained from all the parents.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eLiu KE, Hartman M, Hartman A. Management of thin endometrium in assisted reproduction: a clinical practice guideline from the Canadian Fertility and Andrology Society. Reprod Biomed Online. 2019;39(1):49\u0026ndash;62.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSalman MM, Zaki AM, El-Gamal HH, et al. Effect of intrauterine infusion of autologous platelet-rich plasma in patients with refractory thin endometrium undergoing in vitro fertilization. Prz Menopauzalny. 2023;22(2):77\u0026ndash;82.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiao Z, Liu C, Cai L, et al. The Effect of Endometrial Thickness on Pregnancy, Maternal, and Perinatal Outcomes of Women in Fresh Cycles After IVF/ICSI: A Systematic Review and Meta-Analysis. Front Endocrinol (Lausanne). 2022;12:814648.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePirtea P, Cicinelli E, De Nola R, et al. Endometrial causes of recurrent pregnancy losses: endometriosis, adenomyosis, and chronic endometritis. Fertil Steril. 2021;115(3):546\u0026ndash;60.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi W, Cao Z, Yu X, et al. Effect of growth hormone on thin endometrium via intrauterine infusion. Ann Transl Med. 2021;9(16):1325.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSeetho S, Kongwattanakul K, Saksiriwuttho P, et al. Epidemiology and factors associated with preterm births in multiple pregnancy: a retrospective cohort study. BMC Pregnancy Childbirth. 2023;23(1):872.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSunderam S, Kissin DM, Zhang Y, et al. Assisted Reproductive Technology Surveillance - United States, 2018. MMWR Surveill Summ. 2022;71(4):1\u0026ndash;19.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJin H, Han Y, Zenker J. Cellular mechanisms of monozygotic twinning: clues from assisted reproduction. Hum Reprod Update. 2024;30(6):692\u0026ndash;705.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNakasuji T, Saito H, Araki R, et al. The incidence of monozygotic twinning in assisted reproductive technology: analysis based on results from the 2010 Japanese ART national registry. J Assist Reprod Genet. 2014;31(7):803\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eScaravelli G, Pisaturo V, Levi Setti PE, et al. Monozygotic twin rate among ART centers: a multicenter analysis of data from 18 Italian units. J Assist Reprod Genet. 2022;39(10):2349\u0026ndash;54.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZou G, Ji Q, Chen J, et al. Perinatal outcome and timing of selective fetal reduction in dichorionic diamniotic twin pregnancies: a single-center retrospective study. Front Med (Lausanne). 2024;10:1327191.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCai P, Ouyang Y, Gong F, et al. Pregnancy outcomes of dichorionic triamniotic triplet pregnancies after in vitro fertilization-embryo transfer: multifoetal pregnancy reduction versus expectant management. BMC Pregnancy Childbirth. 2020;20(1):165.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMeireson E, De Rycke L, Bijnens EM, et al. Birth outcomes of twins after multifetal pregnancy reduction compared with primary twins. Am J Obstet Gynecol MFM. 2024;6(1):101230.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLu Y, Tian T, Chen L, et al. Diverse impacts of female chromosomal polymorphisms on assisted reproduction outcomes: a retrospective cohort study. BMC Pregnancy Childbirth. 2024;24(1):331.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXie X, Li F, Tan W, et al. Analysis of the clinical features of pericentric inversion of chromosome 9. J Int Med Res. 2020;48(9):300060520957820.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-pregnancy-and-childbirth","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"prch","sideBox":"Learn more about [BMC Pregnancy and Childbirth](http://bmcpregnancychildbirth.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/prch/default.aspx","title":"BMC Pregnancy and Childbirth","twitterHandle":"@BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Thin endometrium, Multiple pregnancy, Frozen-thawed embryo transfer, Natural reduction of tire","lastPublishedDoi":"10.21203/rs.3.rs-8428992/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8428992/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEndometrial thickness is a key index to evaluate endometrial receptivity, which is closely related to embryo implantation and pregnancy outcome. Thin or damaged endometrium can lead to embryo implantation failure and early pregnancy abortion. It is extremely rare for patients with thin endometrium to have multiple pregnancies.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCase presentation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA 35-year-old woman with primary infertility for 1 year, thin endometrium caused by chronic endometritis, and combined with a variety of other infertility factors. In the fresh cycle, 8 oocytes were obtained by short-acting long protocol, and 7 embryos were frozen. The fresh cycle transplantation was cancelled due to insufficient endometrial thickness (6.7 mm). The endometrial microenvironment was improved by comprehensive treatment during the resuscitation cycle. After the endometrium was increased to 8.3 mm, the single blastocyst was resuscitated and transplanted, and the triplet pregnancy of double chorionic triple amniotic sac was obtained. After natural reduction, a healthy baby boy was delivered at term.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEven if single blastocyst transplantation, there is still an unpredictable risk of single oocyte division, which can lead to high-order multiple pregnancy. Clinically, patients should be fully informed of the risk, and closely monitored during early pregnancy. If necessary, selective reduction should be performed in time to improve maternal and infant outcomes.\u003c/p\u003e","manuscriptTitle":"A case report of twin-chorionic triamniotic triplet pregnancy following single blastocyst transfer in a thin endometrium","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-23 00:38:37","doi":"10.21203/rs.3.rs-8428992/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2026-01-28T10:47:39+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"24644666836835299223226378282753494086","date":"2026-01-25T18:44:40+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"267563550210792735573680628557858349646","date":"2026-01-21T10:34:11+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-01-20T18:18:57+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-01-19T10:12:58+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-12-29T18:35:05+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-12-24T15:26:58+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Pregnancy and Childbirth","date":"2025-12-24T15:20:17+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-pregnancy-and-childbirth","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"prch","sideBox":"Learn more about [BMC Pregnancy and Childbirth](http://bmcpregnancychildbirth.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/prch/default.aspx","title":"BMC Pregnancy and Childbirth","twitterHandle":"@BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a3215011-ea36-4b9a-ac0a-2274a73bb7a5","owner":[],"postedDate":"January 23rd, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-01-23T00:38:39+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-23 00:38:37","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8428992","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8428992","identity":"rs-8428992","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}