Improvement in Th1/Th2 balance and endometrial receptivity after hysteroscopic surgery in patients with cesarean scar disorder: a case report.

A 37-year-old G1P1 woman with a history of cesarean section at 24-year-old and laparoscopic myomectomy at 30-year-old was referred to our clinic for the treatment of secondary infertility. The patient had undergone unsuccessful intrauterine insemination six times at a referral clinic. After the cesarean section, her menstrual period became longer, spotting was unpredictable, and pain was worsened. She felt stressed because of her symptoms. Ultrasonography revealed a CSD with a small collection of IUF (Fig. 2 -A). She received ART and a frozen embryo transfer (FET) in the hormone replacement cycle (HRC) for the first time (5AB by the Gardner classification), but she did not get pregnant. After the first failed transfer, the patient underwent endometrial biopsy for endometrial microbiome metagenomic analysis (EMMA; Igenomics, Japan), Analysis of Infectious Chronic Endometritis (ALICE; Igenomics, Japan), and CE with immunohistochemical staining using the CD138 antibody. The examination revealed a non-Lactobacillus dominant (ultralow) environment and no CE (CD138 positive cell were zero). Second (4BB), third (5BB and 4CC), and fourth (3AB) FETs during the HRC were attempted after vaginal Lactobacillus probiotic treatment, which failed and resulted in a diagnosis of repeated implantation failure (RIF). Because the patient had bilateral hydrosalpinx with IUF, she underwent laparoscopic bilateral fallopian tube resection. Although surgery was performed, a small amount of IUF remained. The patient underwent further examinations as follows: hysteroscopy revealed the presence of a hyper-vascularized area and dendritic vessels in the CSD (Fig.  1 ), ERA resulted in pre-receptive (117 ± 3 hours), EMMA was improved but remained non-Lactobacillus dominant (79.2%) without CE (CD138 positive cell were zero), and the Th1/Th2 ratio was 25.6 (Th1 20.5% and Th2 0.8% during the luteal phase). A fifth (4BB) FET was attempted with vaginal Lactobacillus probiotic treatment, personalized FET according to the ERA, and 3 mg of tacrolimus. Regardless of these treatments, FET did not lead to pregnancy. The patient’s symptoms satisfied the diagnostic criteria for CSDi [ 1 ], and she finally decided to undergo hysteroscopic surgery for the CSD. Measurements of isthmocele size were performed in the early luteal phase of the menstrual cycle. Isthmocele volume approximated as a pole triangle: estimated volume (depth x length x height/2). Magnetic resonance imaging before myomectomy revealed a residual myometrium thickness (RMT) of 4.0 mm and an isthmocele volume of 891.75 mm 3 (Fig.  2 -B). Hysteroscopic surgery was performed under general anesthesia and laparoscopic observation. The surgery was performed as follows: the inferior edge of the CSD was resected with a cutting loop electrode, and the dendritic vessels at the bottom of the CSD were cauterized. Three months postoperatively, magnetic resonance imaging revealed an RMT of 7.9 mm and an isthmocele volume of 522.4 mm 3 (Fig.  2 -C). The symptoms of CSDi, such as abnormal spotting, intrauterine fluid, and pain were improved. In addition, the ERA test became receptive and the Th1/Th2 ratio was decreased to 10.8 (Th1 23.7% and Th2 2.2% during the luteal phase) postoperatively. Five months after the surgery, a sixth FET (5CB and 4CB) was attempted, which led to a successful pregnancy. At 37 weeks of gestation, healthy male baby weighing 2,784 d was born by cesarean section (Timeline of treatment is Fig. 3 ). Fig. 1 Hysteroscopic finding of the bottom of the CSD. Hypervascularization and dendritic vessels were observed Hysteroscopic finding of the bottom of the CSD. Hypervascularization and dendritic vessels were observed Fig. 2 A : Sagittal view of sonography before operation. B : Sagittal view of T2-weighted magnetic resonance image before myomectomy. C : Sagittal view of T2-weighted magnetic resonance image after operation. Arrowheads: cesarean scar defect (isthmocele). Long arrow: intrauterine fluid. Bidirectional arrow: residual myometrium thickness A : Sagittal view of sonography before operation. B : Sagittal view of T2-weighted magnetic resonance image before myomectomy. C : Sagittal view of T2-weighted magnetic resonance image after operation. Arrowheads: cesarean scar defect (isthmocele). Long arrow: intrauterine fluid. Bidirectional arrow: residual myometrium thickness Fig. 3 Timeline of treatment and test results Timeline of treatment and test results

Blood samples were collected during mid-luteal phase. Immunological tests assessed IFN- γ- producing Th cell (Th1 cell; CD4 + T lymphocytes with IFN- γ without IL- 4) levels, IL- 4- producing Th cell (Th2 cell; CD4 + T lymphocytes with IL- 4 without IFN- γ) levels.12,24 As for Th cell levels, blood samples were analyzed on the sampling day by laser flow cytometry (Fascinator II; BD Biosciences) using Phorbol 12- Myristate 13 Acetate, Ionomycin, Brefeldin- A (Sigma- Aldrich Corp.), CD4 R- phycoerythrin- cyanine (PC)- 5 (Immunotec), Fluorescence- activated cell sorting (FACS) Lysing Solution (BD Biosciences), FACS Permeabilizing Solution 2 (BD Biosciences), FastImmune IFN-, and Fluorescein isothio cyanate (FITC)/IL- 4 PE (BD Biosciences). After surface staining of the activated whole blood samples with anti- CD4– PC5- conjugated mono clonal antibodies, red blood cell lysis and specific intracellular staining using FastImmuneTM IFN- FITC/IL- 4- PE (Becton Dickinson Biosciences) were subsequently performed according to the manufacturer’s instructions. The ratio of IFN- γ- positive to IL- 4- positive Th cell levels was calculated as the Th1/Th2 cell ratio. Th1/Th2 cell ratio > 11.8 was considered to be high [ 10 ].

Cesarean section is a common and essential surgery used to save mothers and newborns. However, there is great concern regarding secondary infertility after cesarean section, known as cesarean scar disorder (CSDi) [ 1 ]. A recent meta-analysis showed that isthmoceles negatively affect assisted reproductive technology (ART) outcomes [ 2 ]. The sonographic findings of the uterine niche, sometimes described as a cesarean scar defect (CSD) or an isthmocele, were defined by the European Niche Task Force [ 3 ]. CSD have been reported to exist in 24 to 70% of cesarean sections examined using transvaginal ultrasound [ 4 ]. Approximately 30 to 40% of women with CSDs experience symptoms such as postmenstrual spotting, dysmenorrhea, chronic pelvic pain, and infertility [ 1 ]. These symptoms affect a patient’s quality of life and cause secondary infertility. Vissers hypothesized the following mechanisms of CSD and infertility: CSD-related accumulation of intrauterine fluid (IUF) impairs implantation, alters immunobiology and/or increases inflammation; fibrosis or interruption of the myometrial layer at the site of CSD causes distorted contractility of the uterus; accumulation of mucus and old blood in the CSD may impair sperm penetration; a large CSD in combination with a strongly retroflexed uterus impairs accessibility for an eventual embryo transfer due to a distorted anatomy; CSD-related gynecological symptoms may interfere with sexual intercourse; and focused therapies for niche complaints may interfere with opportunities to conceive during the convalescence period [ 5 ]. One recent study revealed increased levels of inflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, and IL-6 in the pelvic fluid in CSDi patients when compared with controls [ 6 , 7 ]. There is no doubt that CSDi alters localized immunology and increases inflammation, but the underlying mechanism is still unclear. TNF-α is produced from helper T cells (Th1), so it is hypothesized that Th1 can increase in CSDi patients. However, there are no reports on the relationship between Th1/Th2 ratio and CSDi to date. Another study reported the relationship between chronic endometritis (CE) and the window of implantation (WOI) using endometrial receptivity analysis (ERA) [ 8 ], CE, and CSD [ 9 ]. However, no reports have described the WOI or CSD without CE. Many studies have reported that surgical treatment improves pregnancy rates [ 4 ]. In the present case, the Th1/Th2 ratio and ERA improved after hysteroscopic surgery. This is the first report describing the improvement of Th1/Th2 ratios and ERA in patients with CSDi after hysteroscopic surgery.

CSD causes various symptoms but etiology is not yet clarified. This report provides new insights into the etiology of CSD and first described the relationship between CSDi and Th1/Th2 imbalance and endometrial receptivity. Further research is essential to clarify the etiology and strategy to treat CSDi.

The etiology of CSDi in secondary infertility is diverse and complex. Vissers assumed the following etiology: [ 5 ] The effects on the intrauterine environment, isthmocele, anatomical changes, and psychological stress can decrease the chances of pregnancy in ART cases. CSD have been reported to alter the intrauterine environment due to the IUF, leading to CE [ 7 , 9 ] and changes in the microbiome flora [ 11 ]. IUF has been associated with increased integrin expression in mouse models, which may be associated with endometrial receptivity [ 12 ]. Although this study was conducted to determine the cause of hydrosalpinx, the same hypothesis was established for CSDi. However, there are no reports describing the WOI and CSDi to date. Kuroda reported the relationship between CE and WOI using ERA [ 8 ]. A total of 238 genes were included in the ERA [ 13 ], though inflammation can alter the results. In this case, there was no CE; however, the ERA was not receptive. One possible explanation for this is a change in immunology due to CSDi. A recent study revealed an increase in inflammatory cytokines such as TNF-α, IL-1β, and IL-6 in the pelvic fluid of CSDi patients [ 6 , 7 ]. In fact, the patient’s Th1 (which promotes TNF-α) and Th2 ratio was high preoperatively and had normalized with a normal WOI postoperatively. These facts suggest that CSDi can alter the Th1 and Th2 balance, leading to a change in WOI. It remains unknown why TNF-α and IL-1β are high in CSDi patients. Nobuta proposed that these cytokines originated from the uterine cavity or from the peritoneal cavity in endometriosis [ 7 ]. Additionally, we must consider that menstrual pain and psychological stress can change the Th1/Th2 balance because psychologic causes are included in the diagnostic criteria of CSDi. In fact, psychologic stress was reported to increase pro-inflammatory cytokines such as TNF-α, IL-6, and interferon (IFN)-γ [ 14 ]. The etiology of infertility and CSDi remains unclear owing to their complexity. Recent reports positively support the relationship between microbiota-immunity change and CSD. Changes of Microbiome flora possibly related to local inflammation [ 11 ] which possibly lead to CSD and adenomyosis [ 15 ]. Surgical treatment of CSDi improve the local inflammation and fertility, This is the first report to describe the relationship between CSDi, Th1/Th2 imbalance, and ERA. In this case, various treatment such as microbiome flora treatment, personalized embryo transfer, and Th1/Th2 imbalance treatment with tacrolimus did not lead to successful pregnancy, but improvement in WOI and Th1/Th2 balance after surgical treatment.These multifactorial treatments led to a successful pregnancy. Similar to previous reports [ 16 – 18 ], surgical treatment is one of the best and earliest strategies for the treatment of infertility due to CSDi.

This case consisted of multi-factor so that it is hard to conclude one etiology. Peripheral Th1/Th2 is a surrogate for the endometrial environment, further studies involving more cases are important.