The Effect of Intrauterine Infusion of Peripheral Blood Mononuclear Cells Culture on Subendometrial Blood Flow in Patients Undergoing ICSI Cycles

In recent years increasing evidence proposed that local immune cells at implantation site have largely contributed to embryo implantation. The intrauterine infusion of activated peripheral blood mononuclear cells culture 2 days before embryo transfer can enhance the implantation. One of the methods used to evaluate the endometrial receptivity is by assessing the sub endometrial blood flow. A total of 67 infertile women (30) women receives intrauterine non-invasive insemination of peripheral blood mononuclear cells (PBMC) culture 2 days before embryo transfer representing the PBMC test group, and (37) women without receiving any cell as Non-PBMC group. The cultured PBMC are administered into the uterine cavity of the patients. 2 days later, embryos are transferred into the uterine cavity. Endometrial thickness and sub-endometrial blood flow measurements are taken for all cases on trigger and embryo transfer days. On embryo transfer day there was no significant difference (p = 0.770) in mean endometrial thickness between the PBMC group and Non-PBMC group. There was a significant difference (p< 0.001) in the mean resistive index; the level being lower in the PBMC group. Moreover, there was a significant difference (p< 0.001) in the mean pulsatility index. Regarding all enrolled women, the pregnancy rate of 25.4 %, the rate was higher in the PBMC group in comparison with the Non-PBMC group, 43.3 % versus 10.8 %, respectively and the difference was significant (p = 0.002). The use of PBMC culture can improve sub-endometrial.


Introduction
Having difficulty conceiving a child after 12 months of regular, unprotected sexual contact is a medical and social problem that can be defined as the inability to achieve a clinical pregnancy. Depending on the woman's age, medical and reproductive history, fertility interventions may be initiated in less than one year (Zegers-Hochschild, et al. [1] ). Assisted reproductive technology (ART) is a difficult method to resort to, because it includes surgically removing an oocyte from a woman's ovaries and collecting sperms from her husband, mixing them in a special media in a laboratory, and returning them to the woman's uterus to conceive a child (Neri, et al. [2]) . Implantation, despite the medical and clinical progress made in fertility therapy, is a difficult process in which a foreign embryo has to be accepted by the mother's endometrium.
Implantation failure is still a major human reproduction challenge. The underlying reason of implant failure is obviously multifaceted and cannot be linked to any certain defect.
Uterine abnormalities, alterations in the hormone, immunology and thrombophilia are diverse maternal variables that cause failure of implantation (Maleki-Hajiagha, et al. [3] ).
According to the available literature, different maternal immune cell subsets contribute significantly to embryo implantation and are involved in all procedures necessary for the maintenance and completion of a successful pregnancy (Ghaebi, et al. [4] ). It is widely acknowledged that the endometrium can only allow embryo implantation for a certain period of time, known as the implantation window (Blesa, et al. [5] ). Based on the accumulation of data, it has been demonstrated that local immune cells at the implantation site actively contributed to successful implantation of the embryo. It has been discovered that stimulated peripheral blood mononuclear cells improve the rate of implantation (Fujiwara, et al. [6] ).
The use of high-resolution transvaginal probes made it possible to track endometrial changes during the whole menstrual cycle (Hannan, et al. [7] ). Uterine receptivity is influenced by a variety of factors, the most important of which is uterine perfusion and endometrial perfusion.
It has been discovered that there are differences in uterine perfusion between infertile and fertile women. It has been speculated that poor uterine and endometrial perfusion may be the fundamental cause of failure. Blood flow resistance in the uterine artery and the endometrial area has been shown to be a predictive indication of implantation in ART cycles (Haouzi,et al. [8] (Lessey,BA [9] ).

Inclusion Criteria
Patients with age less than 40 years, BMI less than 30 Kg/m 2 , absence of uterine pathology like hydrosalpinx uterine fibroids and endometriosis, and patients who doesn't have at least one embryo grade 1 were excluded from the study.  (Trew, et al. [11] ). All infertile patients received luteal support using progesterone treatment for 2 weeks in the form of 400 mg vaginal progesterone pessaries (Cyclogest; Actavis ® , UK) once daily until a pregnancy test was achieved (Neri, et al. [12] ). incubator (Memert, Germany) (Madkour, et al. [13] ).

ICSI Protocol
Oocytes were retrieved by aseptic transvaginal ultrasound-guided oocyte aspiration, approximately 34-36 hours after rhCG administration under general anesthesia (Trew, et al. [11] [2] ). Prior to embryo transfer, the developed embryos were graded regarding to the embryo grading system (Scott, et al. [15] ). With this system, each embryo was graded as grade 1, 2 and 3.

Two-Dimensional Power Doppler Ultrasound of Endometrial and Subendometrial Zones
Transvaginal ultrasound scanning was performed to assess endometrial thickness, regularity, and echogenicity, as well as color

Statistical Analysis
Data were collected, summarized, analyzed

Results
The current study included a total of 67 infertile women who were categorized into a PBMC group (n = 30) and a Non-PBMC group       Moreover, there was a highly significant difference in the mean PI (p< 0.001); the level being lower in PBMC group, as shown in Table 4. The mean RI was compared between PBMC and Non-PBMC group at day of embryo transfer, as shown in Figure 1.
Moreover, the mean PI between the groups at day of embryo transfer were compared, as shown in Figure 2. Regarding all enrolled infertile women, the pregnancy rate was 25.4 %. However, the rate was higher in the PBMC group in comparison with the Non-PBMC group, 43.3 % versus 10.8 %, respectively and the difference was highly significant (p=0.002), as shown in Figure 3.

Discussion
To achieve successful implantation, the endometrium must be receptive, the embryo must be normal and functional, the dialogue between maternal and embryonic tissues must be coordinated. According to accumulating data, local immune cells at the implantation site have actively contributed to embryo implantation during the development of the embryo (Fujiwara, et al. [6] ). According to another study, the infusion of mouse PBMC   [29] ).

Conclusions
The