Primary outcome: Wound healing and the cosmetic results.

The primary outcome measure is the wound healing measured by the patient and observer scar assessment scale (POSAS) summary score, which consists of the Patient Scar Assessment Scale (PSAS) score and Observer Scar Assessment Scale (OSAS) score. The POSAS is a reliable and valid scar assessment scale designed for a subjective evaluation of various types of scar formation from the perspectives of both the participant and the clinician [18]. The authors will be using POSAS 3.0 for this study [19].

The Patient Scale includes 17 items, each rated on a 5-point Likert scale ranging from “not at all” to “extremely,” evaluating various aspects of the scar over the past week and at the present moment. These items cover parameters such as scar color, pain, sensitivity, itching, surface texture, elevation, dryness, widening, and other sensory symptoms like burning or tingling. The scale is intended to reflect the patient’s subjective experience of the scar, allowing for a more holistic evaluation of scar quality beyond clinical observation alone. The Observer Scale, completed by a trained clinician, assesses additional morphological and functional characteristics of the scar and contains 9 items. The minimum scores for PSAS and OSAS are 17 and 9, and the maximum scores are 85 and 45, respectively. No validated cut-off values exist for POSAS 3.0. It is explicitly intended as a continuous outcome measure for scar quality (higher = worse scar), not a diagnostic test.

The Persian version of the POSAS was translated, culturally adapted, and validated by Naghizadeh et al. in a study of 60 post-surgical patients. It demonstrated strong content validity, high internal consistency (Cronbach’s alpha: 0.88 for the Observer Scale and 0.81 for the Patient Scale), and good test-retest reliability, confirming its suitability for clinical and research use in Iran [20].

Participants and a general practitioner (or an obstetrician and gynecologist) complete the scales at 10 and 28 days after surgery.

Surgical Site Infection (SSI).

We defined the SSI by the Centers for Disease Control and Prevention (CDC) guideline for surgical site infection events [21]. The participants will be visited by a general practitioner (or an obstetrician and gynecologist) for purulent drainage from the superficial incision, localized pain or tenderness, localized swelling, erythema, or heat. The specimen is obtained aseptically from confirmed cases of the SSI and is sent to the laboratory for microbiological testing. Patients with SSI are categorized as superficial incisional SSI, deep incisional SSI, and organ/space SSI based on the mentioned guidelines. Incidence of the SSI is assessed on 36 hours, 10, and 28 days postoperative.

Pain.

Surgical wound pain is assessed using a visual analog scale (VAS) ranging from one to ten. A general practitioner (or an obstetrician and gynecologist) asks the participant about her pain at the four, 12, 24, 36 hours, 10, 28 days postoperative. Also, analgesic medication use by name of the medication, route of intake (oral, intramuscular, intravenous), dosage, and frequency are recorded at the aforementioned time steps.

Other surgical site complications.

All the participants are assessed by a general practitioner (or an obstetrician and gynecologist) at 36 hours, 10, and 28 days postoperative for surgical wound dehiscence, seroma, hematoma, and bleeding.

Safety and adverse event reporting.

All participants are monitored intraoperatively and postoperatively for any signs of adverse events, including but not limited to allergic and immunologic reactions. Any unexpected or serious adverse events are documented and reported in accordance with institutional protocols. In cases of suspected allergic or immunologic reactions, immediate clinical evaluation and appropriate management will be initiated. All adverse events are recorded in the case report forms (CRFs) and reviewed by an independent safety officer not involved in the study procedures.

Recruitment and baseline assessment.

All the participants are recruited from clients referred to Arash Women’s Hospital. The researchers recruit participants through in-person explanation, and interested clients receive detailed information about the study. After checking that the eligibility criteria have been met, a written informed consent will be signed by the participant. The baseline assessment involves demographic characteristics (age, weight, height, and nationality), socioeconomic status (education and place of residence), and medical history and conditions (parity, number of former CS, fasting blood sugar (FBS), two hours oral glucose tolerance test (OGTT), complete blood count (CBC) test, vital signs (VT), and history of underlying diseases and comorbidities) of the participants. All baseline assessments will be conducted by trained medical residents.

Eligibility criteria.

All women aged 18–45 years, singleton pregnancy, having a previous plan for CS and elective CS, planned gestational age of at least completed 36 weeks (≥37 + 0 weeks) at the time of delivery, with hemoglobin (Hb) above 10 g/dL at the time of hospitalization, able to provide the consent, and with intention to be available for the entire research period and to complete all relevant study steps, including follow-up visits and phone calls are included in the study.

The researchers exclude all participants with a body mass index (BMI) greater than or equal to 40 at the time of entering the study, placenta previa or placenta accreta, history of intestinal or urological surgery, multiple pregnancy, known or suspected immunodeficiency (including HIV, hepatitis B or C infection), use of immunosuppressive drugs, chemotherapy, radiotherapy, or taking systemic corticosteroids in the 4 weeks before surgery, known tobacco or drug use, history of keloid formation, participation in another clinical trial in the last three months, any serious cardiovascular, pulmonary, hepatic, renal, digestive, hematologic, endocrine, metabolic, neurological and psychological diseases, frequent vaginal examinations, emergency CS, chorioamnionitis or other systemic infections at the time of presentation for CS, including evidence of subabdominal skin infection (e.g., fungal infection), and any condition that in the opinion of the investigator may pose a risk to the health of the participant or interfere with the evaluation of the study participants.

Intervention.

The participants are divided into four groups:

• Intervention group A: dressing with autologous AM for CS wound.
• Intervention group B: dressing with autologous AF spray for CS wound.
• Intervention group C: dressing with autologous AM and AF spray for CS wound.
• Control group D: simple dressing for CS wound (standard of care).

For all participants in intervention group A, after the surgery, a portion of the AM corresponding to the size of the surgical incision is removed, washed with sterile normal saline, and placed on the incision site before applying the usual dressing. The autologous AM is stored in a sterile receiver filled with sterile normal saline at all times between harvest and application. The storage duration varies depending on the length of the surgery, typically not exceeding 30–60 minutes, and the storage temperature is maintained at room temperature (approximately 20–25°C). In intervention group B, the extracted autologous AF is sprayed directly into the skin incision immediately before wound closure. In group C, both interventions are applied. In the control group D, subjects receive standard care with a simple dressing. Wound closure was standardized across all groups and performed using absorbable subcutaneous sutures followed by non-absorbable interrupted sutures for the skin, applied by the same surgical team following a consistent technique. During the surgery, after the uterine incision, the following steps are performed for groups B and C:

1. After incising the lower part of the uterus, the amniotic sac is opened with an amniotic perforator.
2. A 10 ml syringe is used to drain the AF.
3. Then, the 10 ml syringe containing the AF is observed for contamination (for example, the presence of meconium), and then the AF is sprayed throughout the skin incision (1.5 to 2.0 ml).
4. Then, the cut is dressed as usual.

Randomization.

To make sure that the same number of participants is allocated to all four groups, block randomization with block sizes of 4, 8, and 12 is used. A randomized list is generated using the Sealed Envelope website [22]. The number of blocks and the allocation sequence in the randomized list are only available to an external observer responsible for participant allocation, and the researchers are not aware of it. This external observer will not have a role in analyzing the data, assessing, or evaluating the participants. Adherence to the randomization protocol will be closely monitored throughout the trial.

Blinding and study termination.

The wound dressings of all four groups are similar from the outside, and since the AM is placed under the dressing, the subject will not be aware of her allocation. Blinding of the surgical team is not possible. The physician who examines the condition of the wound is not aware of the participant’s group, and the participant’s wound dressing is opened by another physician or nurse before the examination by the physician, so that the examining physician does not know about the assignment. In general, except for the surgical team and the external observer, all participants involved in the assessment and analysis of the study will be blinded.

The process of unblinding and/or discontinuing a participant will be done only in case of necessity and the occurrence of a serious side effect (with the diagnosis and order of the supervisor of the study). The blinding of the study will be broken only in cases where it is not possible to manage and treat the participant appropriately and adequately without knowing the type of intervention she received.

Concealment.

Sequentially numbered, opaque, sealed envelopes are used for concealment. Each envelope is identical, fully opaque to prevent visibility of contents, and sealed to prevent tampering. Envelopes are stored in a locked cabinet and are only accessible to an independent research assistant not involved in participant recruitment or assessment. Envelopes are opened sequentially only after confirming participant eligibility and obtaining informed consent.