Study the Effects of Intrauterine Flushing With Low Dose Human Chorionic Gonadotropin on Ultrasound Parameter and Immunological Marker of Endometrial Receptivity of Infertile Female Undergoing ICSI Cycle
Phase: PHASE4
Conditions: Female Infertility
Sponsor: Omayma Ismail Khalaf

 
Study the Effects of Intrauterine flushing with Low Dose
Human Chorionic Gonadotrophin on Endometrial 
Receptivity and ICSI Outcome 
OCTOBER 16, 2024 
   

Chapter Three Patients, Materials and Methods 
A prospective comparative study was conducted in the High Institute for 
Infertility Diagnosis and Assisted Reproductive Technologies, Al-Nahrain 
University in Baghdad, from January 2023 to January 2024. Ninety Iraqi women 
were included in this study. Every patient gave her written informed consent before 
taking part in the study figure (3-1), which was approved by the Ethical Committee 
of High Institute for Infertility Diagnosis and Assisted reproductive Technologies, 
Al-Nahrain University. They were evaluated twice during the study period and they 
did not receive any monetary compensation for their participation.  
3.1. Patients  
A study involved a total of ninety women who were selected from those 
attended the High Institute for Infertility Diagnosis and Assisted Reproductive 
Technologies.  
3.1.1. Patient classification 
 Group A: patients who receive (700IU) of intrauterine hCG  
 Group B: patients who receive (500IU) of intrauterine hCG 
 Group C: Control group patients did not receive any treatment. 
3.1.2. Inclusion criteria  
• All couples undergoing IVF / ICSI protocols reaching the day of embryo transfer 
with grade 1 (G1) embryos.  
• Age of women was (18-40) years old.  
• Infertility due to female factors: tubal blockage, un ovulatory cycles, unexplained 
infertility. 

• Infertility due to male factors. 
• fresh cycle transfer 
3.1.3. Exclusion criteria  
• All cases of endometrioses whether suspected clinically or diagnosed 
laparoscopically. 
 • Cases of congenital anomalies of the reproductive system.  
• Patients aged more than 40 year 
 • Those not having G1 embryos.  
• Cases with chronic systemic diseases (diabetes, blood disease, hypertension, 
connective tissue and autoimmune diseases, asthma, class II and III obesity, thyroid 
disturbance).   
3.2 Materials equipment and tools 
3.2.1. Equipment  
The current study utilizes the tools, instruments, chemical substances and 
kits, outlined in Tables (3-1), (3-2) and (3-3). 
 
Table (3-1) Apparatus and Equipment 
          Apparatus & Equipment 
        Company 
Country 
 
Air Incubator 
Memmert 
Germany 
Automatic Pipette 
Slammed                          
Germany 
Centrifuge  
Eppendorf  
Germany 
CO2 Incubator 
Galaxy47/Eppendorf/ 
Germany 
Collecting Tubes 1.5 ml 
AFCO 
Jordan 

Dry Incubator 
Fisher scientific 
USA 
EZ- tip RI 
Cooper surgical fertility 
companies, Malov 
Denmark 
EZ-grip RI 
Cooper surgical fertility 
companies, Malov 
/Denmark 
Flexible 
catheter 
(embryo 
transfer 
catheter 
Gynetics 
 
Ireland 
Freezer 
Concord 
lebanon 
Gel &Clot Activator Tubes 
AFCO 
Jordon 
Glass Pasteur Pipette(150mm) 
Poulten & Graf LTD 
England 
ICSI manipulator 
Eppendorf/Nikon Te2000- 
4 
Germany 
IUI catheter 
Gyntics 
Beligum 
Laminar Flow Cabinet 
K-Systems 
Denmark 
Light microscope 
MEIJI 
Japan 
Medical suction machine 
ARDO 
Switzerland 
Micropipette of 5-50, 100-1000μl 
Thermo Scientific Nunc IVF 
Product 
Denmark 
Microplate reader capable of measuring 
absorbance at 450 nm 
Human reader 
Germany 
Mini vidas Automated Immunoassay 
BIOMRIEUX/ 
Italy 
Ovum aspiration needle 
Cook® 
Australia 
Pasteur pipette and tubes 
Eppendorf 
Germany 
Pipette Tips (Yellow, Blue) 
Brand  
USA 

Pipette Tips (Yellow) 
Brand 
Germany 
Plain Tubes 
AFCO 
Jordon 
Plastic rack 
AFCO  
Jordon 
RI holding pipette 
Cooper surgical fertility 
companies, Malov 
/ Denmark 
RI Spiked Injection Pipette 
Cooper surgical fertility 
companies,Malov 
Denmark 
Semi-automatic 
microprocessor 
controlled photometer 
Humalyzer Primus 
/Germany 
Scale (weight &height measure) 
Health scale  
China 
Slides and cover slips 
 
Germany 
Stereomicroscope 
Olympus/SZx16 
Japan 
 Syringes (5ml, BD DiscarditTMII) 
Becton Dickinson 
USA 
Test tubes, Falcon petridish, Small dish, 
Center well dish, Four well dish, ICSI 
dish 
Thermo Scientific Nunc IVF 
Product 
Denmark 
Vaginal Sonography 
Vesana Balance 
china 
Wash bottle 
Brand  
India 
 
 
 
3.2.2. Chemicals and drugs 
The chemicals and drugs that used in this study are summarized in Tables (3.3 - 
3.4). 

Table 3.2: Chemicals and their manufactures 
 
Table (3-3): Drugs used in ICSI and their manufactures : Antagonist protocol  
 
 
 
 
 
 
 
 
 
Table 
3.4 
Che
mical
s and 
kits 
 
Chemicals 
Manufacturing 
company/Country 
Ferticult TM Aspiration 
FertiPro NV/Belgium 
FerticultTM Flushing medium 
FertiPro NV/Belgium 
FerticultTM Mineral oil 
FertiPro NV/Belgium 
Gain medium/ Early 
FertiPro NV/Belgium 
Hyaluronidase medium 80IU/ml 
FertiPro NV/Belgium 
PVP 10% in Flushing medium 
FertiPro NV/Belgium 
Trade Name 
Chemical name 
Company & country 
 
Gonal f 
Recombinant FSH 75u injection 
Merk, Switzerland 
 
Cetrotide® 
Cetrorelix acetate for injection 0.25 mg 
Merk, Switzerland 
 
Oviltrelle 
250 microgram hCG 
Merk, Switzerland 
 
Cyclogest 
Natural   Progesterone 
CoxPharmaceuticals
,                                     
Barnstaple, UK 
hCG 
Urinary human chorionic gonadotropin 
Diclaire  
Triptorelin  
(Decapeptyl®) 0.1 mg vial 
 
Ferring/ Germany 

3.3. Methods 
3.3.1. Patients’ assessment and study design  
A comprehensive medical, surgical, and obstetrical history was gathered 
from couples experiencing infertility, as per a structured questionnaire illustrated in 
figure (3-2). Thorough examinations were conducted on infertile women, 
encompassing both general and gynecological assessments. Height and weight 
measurements were taken to calculate the body mass index (BMI) using the 
formula BMI = weight (kg) / [height (m)]2. Initial hormonal evaluations were 
performed on either the second or third day of the menstrual cycle for all female 
subjects. Procedures such as transvaginal ultrasound, saline infusion sonography 
(SIS), and hysterosalpingography (HSG) were carried out to evaluate the uterine 
cavity, confirm tubal patency, and in some cases, laparoscopy was employed to 
assess tubal patency and exclude pelvic abnormalities. Seminal fluid analysis was 
conducted for the male partners. All infertile women enrolled underwent the 
application of the Gonadotropin Releasing Hormone (GnRH) antagonist ovarian 
hyperstimulation protocol, known as the flexible protocol. The follow-up of 
Chemicals and Kits  
Company 
Country 
Human TNF Kit and IL_10 ELISA 
Kit 
R&D Systems biotechne`brand 
USA 
Liaison FSH kit 
Liaison 
Italy 
Liaison LH kit 
Liaison 
Italy 
Liaison TSH kit 
Liaison 
Italy 
Liaison PRL kit  
Liaison 
Italy 
TOSOH E2 Kit 
TOSOH AIA 600II 
Japan 

patients included serial vaginal ultrasound and monitoring of serum estradiol 
levels. Subsequently, ovum pick up (OPU) was carried out post ovulation 
triggering approximately 35-36 hours later. Doppler ultrasound, along with 
measuring serum TNF and IL-10 levels, was performed for all participants on the 
day of OPU, followed by additional measurements on the day of embryo transfer. 
Evaluation of oocytes' morphology and recording of maturation rate (MR) were 
conducted, leading to Intracytoplasmic Sperm Injection (ICSI) of apparently 
normal Metaphase II (MII) oocytes. The morphological assessment of embryos 
was done, and the count of different embryo grades was noted. Luteal phase 
support, embryo transfer, and serum beta-human chorionic gonadotropin (B-hCG) 
evaluation were done on day 14 post embryo transfer, with the recording of 
biochemical pregnancy rate (Figure 3.3). 
 
 
 
 
 
 
 
 
 
 
 
 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
figure (3-2): Questionnaire 
 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
   
 
 
 
 
 

3.3.2. Anthropometric measurement  
            The anthropometric parameters examined in the research primarily 
encompassed Body Mass Index (BMI), as determined by the subsequent criteria.  
The computation of BMI involves dividing the weight in kilograms by the square 
of the height in meters (kg/m2). Subsequently, in accordance with WHO standards, 
females are categorized based on their BMI as follows: 
 those with a BMI ≤18.5 are classified as underweight, 
 individuals with a BMI of 18.5-24.9 are considered to have a normal weight,  
those falling within the 25-29.9 range are classified as overweight, and 
 those with a BMI ≥ 30 are categorized as obese. The formula for calculating BMI 
is expressed as BMI = Weight in Kg ÷ (height in m)2. 
3.3.3. Basal fertility workup  
 Clinical and gynecological examinations were conducted along with basal 
ultrasound scanning via transvaginal route in order to rule out any potential 
abnormalities.  
 Baseline hormonal evaluation was carried out during the early follicular 
phase (day 2 or 3 of the menstrual cycle) encompassing Follicle-
Stimulating Hormone (FSH), luteinizing hormone (LH), and estradiol (E2). 
The assessment of hormones was executed employing an enzyme-linked 
fluorescent assay (ELFA) method, utilizing Minivids analysis apparatus 
(BIOMRIEUX/France). 
 Viral studies were conducted for both partners. 
3.3.4. Intracytoplasmic sperm injection program  

3.3.4.1.a. The flexible antagonist Protocol  
              A flexible antagonist protocol type was implemented for ICSI across all 
participants, with ovarian stimulation commencing once all parameters were within 
normal ranges. These parameters included an endometrium thickness of less than 5 
mm, absence of any follicle exceeding 10 mm in the ovaries, and E2 levels below 
50 pg/ml. The protocol entailed ovarian stimulation through daily subcutaneous 
injections of rFSH (Gonal F, Merk Serono)®, each containing 75IU per ampoule. 
The initial gonadotropin dosage was tailored based on individual factors such as 
age, antral follicle count, BMI, and previous responses to ovulation induction. 
Administration began on the second day of the menstrual cycle. Additionally, some 
participants were administered HMG vials (Menogon, Ferring Pharmaceutical) 
intramuscularly, each containing 75 IU of FSH and LH per vial, in accordance with 
their clinical needs. To address patient-specific requirements, a flexible regimen of 
GnRH antagonist (Cetrorelix acetate 0.25 mg, s/c Cetrotide®, Merck, Switzerland) 
was initiated once the primary follicle reached 13-14 mm. Serum E2 levels were 
also 
monitored 
throughout 
the 
procedure.                  
3.3.4.1.b.Triggering 
of 
ovulation                     
Induction of final oocyte maturation occurred when a minimum of three dominant 
follicles reached a size equal to or greater than 17–18 mm in diameter through the 
administration of subcutaneous recombinant hCG (Ovitrelle®, 6500 IU Merk 
serono, Switzerland), or initiation was facilitated by the use of a "dual trigger" 
comprising human chorionic gonadotropin (hCG) in combination with 
gonadotropin releasing hormone agonist (GnRH-a), specifically triptorelin 
(Decapeptyl; Ferring Pharmaceuticals) administered subcutaneously (. Le MT, et al. 
2019) 
 

3.3.4.2. Transvaginal ultrasound guided oocyte retrieval  
A specialist recovered the oocytes 34–36 hours post-triggering, just prior to 
follicular rupture. Patients were admitted to the operating theatre in a fasting state, 
positioned lithotomy with an empty bladder, administered general anesthesia, and 
subjected to vaginal lavage with normal saline. Oocytes were retrieved from 
individuals under transvaginal ultrasound guidance using a delicate single lumen 
aspiration needle (Gynetics®, Belgium) connected to a Wallace oocyte recovery 
system. The opposite end of the needle was linked to a suction apparatus operating 
at 120-130 mm Hg. (Atzmon et al., 2020). Every follicle in both ovaries was 
aspirated and promptly transferred to the embryologist for meticulous examination 
to ascertain the constitution of the collected cumulus-oocyte complexes. 
Subsequent to completion, the needle was withdrawn, ensuring successful 
hemostasis. The entire procedure typically lasted between 20-30 minutes. 
Subsequently, patients received analgesics, antibiotics, and luteal phase support in 
the form of progesterone (Zheng et al., 2019). 
3.3.4.3. Oocyte grading  
          The categorization of oocytes in terms of their maturity grade was conducted 
subsequent to the removal of the corona layers and cumulus. Subsequently, the 
oocytes underwent a thorough washing process and were examined under an 
inverted microscope to assess their stage of maturity and overall integrity. The 
evaluation of oocyte nuclear maturity relied on the presence of an extruded first 
polar body (PBI) within the perivitelline space and the absence of a germinal 
vesicle (GV). The germinal vesicle (GV) denotes the least mature oocyte where 
this circular structure is situated within the oocyte (refer to Figure 3.4, A). The MI 
stage represents an immature oocyte characterized by the absence of a polar body 
or germinal vesicle, serving as an intermediary phase between GV and MII 

(Coughlan C, Ledger B, 2011) (refer to Figure 3.4, B). MII signifies the most 
mature oocyte containing a polar body (refer to Figure 3.4, C). 
 
 
 
 
 
 
Figure 3.4 depicts oocytes exhibiting varying degrees of maturity; (A) illustrates an oocyte 
classified as Germinal vesicle grade of maturity; (B) showcases an oocyte at Metaphase I grade 
of maturity; and (C) displays a typically mature metaphase II oocyte. This information was 
sourced from the esteemed High Institute for Infertility Diagnosis and Assisted Reproductive 
Technologies at AlNahrain University in Baghdad, Iraq. 
 
According to Ozgur et al. (2015), the maturity index, or the proportion of 
mature oocytes for each participant, was determined using the following formula: 
Maturity index = (Total number of mature oocytes (MII) / Total number of oocytes 
retrieved) × 100. 
 
3.3.4.4. Fertilization and Embryo Culture 
 3.3.4.4.a. Sperms Preparation  
           Usually before OPU, semen sample was prepared for sperm extraction. The 
sample was collected in a private room near the laboratory in order to limit the 
exposure of the semen to fluctuations in temperature and to control the time 
between collection and analysis. The sample was collected after a minimum of 2 
days and a maximum of 7 days of sexual abstinence. The sample was obtained by 

masturbation and ejaculated into a clean, wide-mouthed container made of glass or 
plastic, from a batch that had been confirmed to be non-toxic for spermatozoa. The 
specimen container was kept at ambient temperature between 20 °C and 37 °C to 
avoid large changes in temperature that may affect the spermatozoa after they were 
ejaculated into it. It was labeled with the man’s name, identification number, date 
and time of collection. The specimen container was placed in the incubator (37 °C) 
while the semen liquefied. In men with azoospermia Testicular Sperm Extraction 
(TESE) is a surgical sperm retrieval procedure used as infertility treatment. It was 
done by removing a small portion of tissue from the testicle under general 
anesthesia and extracting the few viable sperm cells presented in that tissue for 
ICSI. 
3.3.4.4. b. Intracytoplasmic sperm injection procedure 
             At the IVF lab, aspirated follicles were immediately examined in a petri 
dish. The oocyte was retained in the CO2 incubator at 37°C for one to two hours 
after flushing. Following that, each oocyte was graded and stripped in the laminar 
flow 
cabinet.  
The mature oocyte was grasped using a specialized holding pipette, and one chosen 
sperm with normal morphology was immobilized and picked up using an ICSI 
microscope and a very thin, pointed, hollow needle. The oocyte's shell was then 
gradually punctured with the needle. (with PB at 12 or 6 o'clock) into its cytoplasm 
(Figure 3.5). To reduce the possibility of chromosomal or spindle damage, the 
genetic material is kept away from the injection site because it is anticipated to be 
adjacent to the PBI. Once the sperm were carefully injected into the cytoplasm and 
the needle was taken out, the sample was kept in a CO2 incubator to await the 
potential result of fertilization. 
 

 
 
 
 
 
 
 
Figure 3.5: ICSI Micromanipulation, Polar body at 12 or 6 o'clock in ICSI images, adapted from 
Al Nahrain University, Baghdad, Iraq's High Institute for Infertility diagnosis and Assisted 
Reproductive Technologies 
3.3.4.5. Evaluation of fertilization and embryo grading :  
Figure 3.6 illustrates the assessment of injected oocytes, which was carried out 16–
18 hours after the ICSI to approve fertilization (Zygotes) and ensure the presence 
of two polar bodies and two pronuclei. If the fertilized oocytes had either 1PN or 
3PN, they were deemed abnormal. It was considered that oocytes lacking PN were 
not fertilized. The following formula was utilized to calculate the fertilization rate: 
The calculation of fertilization rate is (total number of injected MII retrieved 
oocytes / number of fertilized oocytes (2PN) times 100 (Magaton et al., 2023).  
 
 
 
 

 
 
 
 
 
 
 
 
Figure 3.6. Fertilized oocyte (appearance of two pronuclei) is an indicator of     fertilization 
(Merchant et al. 2011) 
 
First-day cleavage was assessed 24 hours after fertilization, and the number 
and size of blastomeres in each embryo were counted. On days 2 and 3 (48 and 72 
hours after oocyte harvest), the amount, size, symmetry, and percentage of 
blastomere fragmentation determine how the embryos are graded. As seen in 
Figures 3.7–3.9, the developing embryos were morphologically graded according 
to the Istanbul consensus workshop prior to embryo transfer, where the embryo 
was classified into grades I, II, and III (Balaban et al., 2011). 
 
 
 
 
 
 
 

 
 
 
 
 
 
 
 
 
 
 
 
       Figure (3-7): Stages of human pre-implantation embryo development. Phase-contrast images of       
human embryo development from day (d) 0 to day 3. Following fertilization, embryos undergo a 
series of mitotic cell divisions. A: Zygote, B: 4 cells Grade 1, C: 8cells Grade 1(High Institute for 
Infertility Diagnosis and Assisted Reproductive Technologies, Al-Nahrain University, Iraq, 
Baghdad, 2023). 
 
Grade II: High quality, most cells have stage-specific cell sizes, even in cell 
division embryos with 10–25% fragmentation. 
 
 
 
 
 
 
 
 
 
Figure 3.8: Adapted from the High Institute for Infertility diagnosis and Assisted Reproductive 
Technologies, Al Nahrain University, Baghdad, Iraq; Day 3 grade 2 embryo 8 cells, equal size, 
20% 
fragmentation 
 
Grade III: Embryos of fair quality that show signs of multinucleation, 
fragmentation (>25%), and uneven cell division. 
 

 
 
 
 
 
 
 
 
Figure 3.9: Adapted from the High Institute for Infertility diagnosis and Assisted Reproductive 
Technologies, Al Nahrain University, Baghdad, Iraq, Day 3 grade 3 embryos (5–6) cells, uneven, 
>25% fragmentation. 
 
3.3.4.6. Embryo Transfer  
Anesthesia was not necessary for the embryo transfer procedure (Orvieto et al., 
2009). Based on the patient's age, clinical history, number of prior tries, and 
embryonic quality, the number of embryos selected for transfer was determined 
(Kamath et al.,2020). Using a flexible catheter (Gynetics®, Belgium) that goes 
through the cervix and the vagina to enter the uterine cavity, fresh embryo transfer 
was performed. The patient was positioned in the lithotomy posture with a partially 
full bladder. To improve the outcome, the cervical mucus was removed and the 
vagina was gently cleaned with normal saline while being guided by trans-
abdominal ultrasound. The highest morphological standards were used in the 
selection of the transferred embryos. The embryologist loaded and deposited the 
embryo into the transfer catheter, which was carefully removed after the embryos 
were transferred to the uterus. The embryo was then examined under a microscope 
to ensure that every embryo had been transplanted. Typically, the embryo transfer 
process takes a few minutes. About 14 days following the embryo transfer, a 
pregnancy test was conducted to ensure the pregnancy was successful. 

3.3.4.7. Luteal Phase Support 
The luteal phase was maintained starting from oocyte retrieval day through 
the administration of vaginal progesterone (Cyclogest®400mg twice daily; 
Actavis, UK®) and progesterone injection (depote 250-500 mg twice weekly) for a 
duration of 14 days. This regimen continued until the β-HCG test was conducted; 
in case of a positive result, progesterone intake was sustained until 9-12 weeks, 
coinciding with the initiation of placental activity (Zhang et al. 2020). 
3.3.5. Samples collection 
3.3.5.1. Blood Sampling 
` 
 Blood Sampling for TNF-α & IL-10, on the day of OPU and day of embryo 
transfer for both hCG group together with control group and according to the 
number and grading of the embryos, patients were prepared for the transfer. The 
patient's serum was obtained by aspirating the blood samples from the median 
cubital vein by disposable syringe (Becton Dickinson company, USA), into a 
serum separating tube (gel & clot activator, AFCO, Jordan), and allowed to clot for 
30 minutes. Serum was obtained after centrifugation at a rate of 5000 rpm for 5 
minutes. All samples were kept in a sterile container labeled with patient’s study 
number. Samples were refrigerated at -20°C until the time of examination using 
diagnostic Kit by (ELIZA) technique (IL-10, TNF, ELIZA Kit, Bio Source, USA). 
3.3.6. Biochemical Assay 
3.3.6.1. Principle of the ELISA: 
 Enzyme-linked immunosorbent assay (ELISA) is a biochemical assay that 
uses antibodies and an enzyme-mediated color change to detect the presence of 
either antigen such as proteins, peptides, and hormones or antibody in a given 
sample (Gan SD.et al., 2013), Figure (3-10) 
 

 
 
 
 
 
 
 
 
           Figure (3-10): Enzyme-linked Immunosorbent Assay (ELISA) (Shan et al., 2002 
 
3.3.6.2. TEST PRINCIPLE FOR IL-10 
This kit uses enzyme-linked immune sorbent assay (ELISA) based on the 
Biotin double antibody sandwich technology to assay the Human Interleukin 10 
(IL-10). Add Interleukin10 (IL-10) to the wells, which are pre-coated with 
Interleukin 10(IL-10) monoclonal antibody and then incubate. After that, add anti 
IL-10 antibodies labeled with biotin to unite with streptavidin-HRP, which forms 
immune complex. Remove unbound enzymes after incubation and washing. Add 
substrate A and B. Then the solution will turn blue and change into yellow with the 
effect of acid. The shades of solution and the concentration of Human Interleukin 
10 (IL-10) are positively correlated figure (3-11). 
 
 
 
 
 
 
 

 
 
 
 
 
 
A 
B 
Figure (3-11): ELIZA washer and shaker 
 
3.3.6.3. TEST PRINCIPLE FOR TNF-α 
This kit uses enzyme-linked immune sorbent assay (ELISA) based on the 
Biotin double antibody sandwich technology to assay the Human Tumor necrosis 
factor-alpha (TNF-alpha). Add Tumor necrosis factor-alpha (TNF-alpha) to the 
wells, which are pre-coated with Tumor necrosis factor-alpha (TNF-alpha) 
monoclonal antibody and then incubate. After that, add anti TNF-alpha antibodies 
labeled with biotin to unite with streptavidin-HRP, which forms immune complex. 
Remove unbound enzymes after incubation and washing. Add substrate A and B. 
Then the solution will turn blue and change into yellow with the effect of acid. The 
shades of solution and the concentration of Human Tumor necrosis factor-alpha 
(TNF-alpha) are positively correlated. 
3.3.6.4. Assay procedure for TNF-alpha: 
  100 μL standard or sample to each well will be added. Incubate for 90 min at 
37℃.  
 The liquid will be removed. 100 μL will be added Biotinylated Detection Ab. 
Incubate for 1 hour at 37℃.  
 Aspiration and wash 3 times. 

  100 μL HRP Conjugate working solution will be added. Incubate for 30 min at 
37℃. 
  Aspiration and wash 5 times. 
  90 μL Substrate Reagent will be added. Incubate for 15 min at 37℃. 
  50 μL Stop Solution will be added. Read at 450 nm immediately.  
 Results will be Calculated. 
3.3.7. Ultrasonography Assessment:  
  3.3.7.1. Endometrial Thickness Measurement  
The transvaginal scan was performed using the 5-9 MHz vaginal probe of 
SonoAceX6 Ultrasound Set, wall filters (50 Hz) were used to eliminate low 
frequency signals, Ultrasound machine was performed after placing patients with 
an empty bladder in the dorsal lithotomy position, a sweep through the mid-sagittal 
plane of the uterus was obtained to give a multiplanar display figure (3-12). 
 
 
 
 
 
 
 
 
 
 
 
 
Figure 3.12: Versana Balance (ge-medical system) ultrasound equipment, CHINA, 
JIANGSU CHINA (High Institute for Infertility Diagnosis and Assisted Reproductive 
Technologies, Al-Nahrain University, Iraq, Baghdad, 2022). 

 
Endometrial thickness of the uterus was measured, as the maximum 
distance from one basal endometrial interface across the endometrial canal to the 
opposite endometrial– myometrial interface, after the patient had completely 
emptied her bladder. The measurement involved both endometrial layers excluding 
the surrounding low amplitude echo layer, three measurements were taken & the 
average value was recorded figure (3-10). 
             
 
 
 
 
 
 
 
 Figure 3.13: Ultrasound review reveals myometrial, endometrial and subendometrial zones 
(Zhao et al., 2012) 
3.3.7.2. Two-Dimensional color doppler Ultrasound 
 
 After completion of the B-mode examination, a pulsed Doppler system 
was used for blood flow analysis. Sub endometrial vessels were visualized at the 
endometrial periphery, sometimes penetrating the hyperechogenic endometrial 
edge or even reaching the endometrial cavity (Elnaggar et al., 2017), The blood 
flow velocity waveforms from the subendometrial vessels were obtained by 
placing the Doppler gate over the colored area at zone 2 and activating the pulsed 
Doppler function. Figure (3-14) 

 
 
 
 
 
      Figure 3.14: A: Subendometrial blood vessels Doppler ultrasound velocimetry in a case  
       of good receptive endometrium. B: Doppler blood flow till Zone 3 
 
A recording was considered satisfactory when at least 3 consecutive 
uniform waveforms were obtained; each demonstrated the maximum Doppler shift. 
Three measurements for each parameter were taken and the average value was  
recorded, Figure (3-12). Resistance index (RI) and pulsatility index (PI) were 
calculated automatically by ultrasound for both subendometrial vessels, according 
to the following formula figure (3-15): 
 
 
 
 
 
 
 
Figure 3.15: three consecutive uniform waveform obtained from doppler study of patient 
attend high institute of infertility diagnosis and assisted reproductive technology. 

  
 
 
 
 
 
 
Figure 3.16: Method of measurement of S/D, RI& PI 
3.3.8. Pregnancy Test (P.T)  
 
Pregnancy is documented by an elevated serum β-hCG level of ≥ 6.5 
mIU/l in the 14th day following embryo transfer, and Progesterone treatment was 
continued up to 12 gestational weeks (Vaisbuch et al., 2012). Ultrasound 
examination is performed 6th week after E.T in order to confirm clinical 
pregnancy, viability and for diagnosis of multiplicity. 
3.3.9. HCG-Intrauterine Infusion 
HCG-intrauterine infusion done for 60 women undergoing ICSI to improve 
endometrial receptivity. Two different doses of hcg (diclair R )1500 IU were used 
for each patient. Drug was used on day of ova pick up.  In the study group, the 
patient was put in the lithotomy position, and the cervix was visualized using 
Cusco’s speculum. The cervical mucous was wiped out using a sterile piece of 
gauze, then the mucous was partially removed by gentle suction with a 1-mL 
syringe. A dummy ET was performed using a soft catheter (Wallace-Smiths 
Medical International Ltd.); if the soft catheter did not easily pass the internal 

cervical os, another more rigid but malleable catheter (Cook IVF) was used. After 
the catheter had passed the internal cervical os.  
3.3.9.1. Preparation of hCG 
HCG dose was prepared by using , 1 ml of tissue culture media (G.2 plus 
ref. 10132, Vitrolife) to one vial containing 1500 IU of hCG (Diclair®). The 
injection dissolved immediately after completion of ova pick up and infused 
directly to endometrial cavity. 35 μL of tissue culture medium containing 500,  or 
47 μL of tissue culture media containing 700 IU of hCG was injected intrauterine. 
Dose prepared by using (1 ml) of gain medium to dissolve hcg injection and dose 
for each patient loaded by using IUI (Gyntics) catheter. 
3.3.9.2. GAIN TM medium  
Single-step culture medium for the in vitro culture of human embryos and 
gametes GAIN™ medium is sterilized by sterile filtration. Medium is a ready to 
use. Medium is a bicarbonate-buffered balanced salt solution with 10 mg/liter 
gentamicin and 3.5 g/liter human serum albumin. GAIN™ medium should 
generally be used in an incubator at 37°C set at 5% CO² at normal atmospheric 
pressure 
 
COMPOSITION GAIN™ medium is a bicarbonate-buffered balanced 
salt solution with 10 mg/liter gentamicin and 3.5 g/liter human serum 
albumin.