The significance of PlGF in the progression of the ovarian hyperstimulation syndrome in patients undergoing an in vitro fertilization procedure.

Placenta growth factor is, VEGF family member, a Pleiotropic Factor affects a variety of cell types and regulates various biological responses. PlGF is similar in structure and function to VEGF, and it amplifies VEGF's angiogenic actions. Ovulation stimulation during cycles of in vitro fertilization (IVF) for the management of infertility results in ovarian hyperstimulation syndrome (OHSS), an iatrogenic side effect. Considering potential roles of PlGF and its receptor (sflt-1) in angiogenesis, the association of these factors with OHSS among study women during controlled ovarian stimulation have evaluated in the present study. Comparative cross-sectional study including of 60 women who go through controlled ovarian stimulation. On oocyte retrieval day, their serum and follicular fluid were taken. The concentrations of PlGF and sFlt-1 were assessed using ELISA .Eighteen patients presented with ovarian hyperstimulation syndrome (OHSS) and 42 patients were no OHSS. There was significantly higher serum PIGF in women with OHSS patients as compared to women with no OHSS, 141.4 ± 11.8 versus 91.9 ± 5.4 respectively (p<0.001). Furthermore, Follicular fluid PlGF there was significantly higher in women with OHSS patients as compared to women with no OHSS, 163.4 ± 7.2 versus 91.1 ± 5.5 respectively (p<0.001). Additionally Follicular fluids PlGF/sFlt ratio significantly higher in women with OHSS patients as compared to women with no OHSS, 0.034 ± 0.01 versus 0.026 ± 0.01 respectively (p<0 . 001). On the contrary there was significantly lower serum and follicular fluid sFlt in ovarian hyper-stimulated patients These statistics show that serum and FF PlGF and PlGF to sFlt ratio higher in women with ovarian hyperstimulation syndrome (OHSS) patients as compared to women with no OHSS. So, the PlGF may be played an important a role in angiogenesis dysregulation


Introduction
PlGF is an angiogenic factor that facilitates the pro-angiogenic action of

Subject material and method
The High Institute of Infertility PlGF and sFlt-1 had sensitivities of 2.54 and 0.25 ng/ml, respectively.

3-Statistical analysis
According to the manufacturer's instructions, this kit uses an enzymelinked immune sorbent assay (ELISA) based on the Biotin double antibody sandwich technique to measure placental growth factor and sFlt-1.

Results
Demographic features between studied groups were showed in (Table

5-Conclusion
These data provide evidence that serum and FF PlGF and PlGF/sFlt ratio is higher in women with ovarian hyperstimulation syndrome (OHSS) patients as compared to women with no OHSS.The serum and follicular fluid sFlt were lower in women with in ovarian hyper-stimulated patients.This gives a suggestion that PlGF may be played an essential task in angiogenic dysregulation.
Ovulation induction during cycles of in vitro fertilization (IVF) results in the iatrogenic condition known as ovarian hyperstimulation syndrome (OHSS).Although OHSS involves 12-25 percent of IVF cycles, severe forms of OHSS occur in 0.5-5 percent of cycles for assisted reproductive technology (ART), and in its critical form, is known to cause maternal death (Naredi, Talwar, and Sandeep, 2014 [1]).The formation of a lot of follicles, elevated estradiol (E2) levels, and larger ovaries are all symptoms of OHSS (Naredi, Talwar, and Sandeep, 2014 [1]).High luteinized bilaterally enlarged cystic ovaries are a hallmark of OHSS.Vascular hyperpermeability, one of the secondary effects, can, in rare cases, be fatal (Nelson, 2017 [2]).The mild type of OHSS's morbidity is probably underestimated because its symptoms may go unrecognized.Moderate OHSS symptoms include abdominal distention, nausea, vomiting, and decreased appetite.Bleeding from an ovarian rupture, acute respiratory distress syndrome, thrombosis, and hepatorenal failure are just a few of the disorders that drive 1.9% of patients to the hospital overall.The critical OHSS could be lethal (Sun et al., 2021 [3]).A rise in capillary permeability, which causes fluid to shift from extravascular to intravascular regions, is a hallmark of OHSS.By rising vascular permeability, VEGF significantly contributes to the pathophysiology of OHSS (Namavar Jahromi B.et al.,2018 [4]).VEGF is generated by the granulosa cells, as well as the human chorionic gonadotropin (hCG), which encourages its production.Higher levels of VEGF are linked to severe OHSS (Namavar Jahromi et al., 2018 [4]).Higher vascular permeability and fluid extravasation are the proposed mechanisms for this condition, which force fluid to leak to enter the lungs and peritoneum, modifications in the The renin-angiotensin system and immunological system have been proposed as pathogenetic precursors to OHSS in addition to enhanced capillary permeability and extensive follicular angiogenesis, both induced by VEGF (Naredi, Talwar, and Sandeep, 2014 [1]).Pro-inflammatory cytokines such as tumor necrosis factor and interleukin-1b, IL-6, and IL-8 have been linked to mediating the immediate phase reaction that results in vascular leakage and the third space loss of OHSS (Naredi, Talwar, and Sandeep, 2014 [1]).These cytokines' serum levels have been observed to be higher in OHSS patients (Naredi, Talwar, and Sandeep, 2014 [1]).

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Diagnosis and Assisted Reproductive Techniques at Al-Nahrain University in Baghdad, Iraq, was where this study took place.The time frame of the study is from October 2021 to September 2022.It is cross-sectional comparative research.Specifically60 women, 30 infertile women with PCOS, and 30 infertile women without PCOS were chosen.Each group was included women that were subjected to ovarian stimulation by antagonist protocol.The indications for Controlled ovarian hyperstimulation in the PCOS group were male factor and tubal factor.Women without PCOS were undergone COS in preparation for ICSI, and these group was matched to the PCOS group by age and BMI prior to enrollment.All involved women provided their written, informed consent.Women were treated using a GnRH antagonist protocol to prevent premature ovulation.Combining recombinant FSH and/or HMG was used to stimulate the ovaries.When there was a possibility of ovarian hyperstimulation or a history of poor response, the standard stimulation procedure was altered.Considering the potential roles of serum and follicular fluid sflt and PlGF in ovarian function, the association of these factors and also PlGF/sFlt-1 ratio with the OHSS by dividing patients according to Rotterdam criteria have been evaluated in the present study.PCOS women were fulfilling two from the three following criteria based on the Rotterdam criteria: Polycystic ovary on ultrasound (at least 12 follicles 2-9 http://doi.org/10.28969/IJEIR.v12.i1.r6.22 et al., Aliyah 73 mm in diameter per ovary), chronic anovulation or oligovulation, clinical or biochemical Hyperandrogenism.The inclusion criteria include infertile women who were diagnosed as PCOS in the presence of at least 2 of the Rotterdam criteria, based on the Rotterdam Consensus Meeting (2003).When at least three follicles are 18 mm with serum E2 measured, two ampules of recombinant hCG 250 mg/0.5 ml prefilled syringe are given and to trigger and induce maturation of the oocyte.Then, 34-36 hours after the hCG injection, oocyte retrieval took place with transvaginal ultrasound guidance under general anesthesia or spinal anesthesia.Embryo transfer was performed transcervical on a patient with a dorsal lithotomy, guided by ultrasound, and the embryo was placed 15-20mm from the fundus.Clinical pregnancy was detected by performing a B-hCG test after two weeks of embryo transfer, followed by a transvaginal ultrasound two weeks later to ensure the viability of the embryo.Collecting and preserving the samples Blood samples and follicular fluid were collected from each woman enrolled in the ICSI procedure.Blood sample was obtained from the patients at cycle day 2. Blood samples was taken for hormone analysis (LH, FSH, E2, testosterone, prolactin) and AMH for infertile women aged 40 years and more assay by MiniVidas.At the day of pickup, the blood sample was taken and allowed to coagulate for 10 minutes, then centrifuged at 3000 rpm for 20 min to separate the serum.The samples were aliquot and quickly frozen and stored at -20C° until assayed.Follicular fluid was collected, and it was utilized for analysis.Following the oocyte's removal, the fluid was centrifuged at 3000 rpm for 10 minutes to take out debris and granulosa cells.Blood or flushing solution were not taken; only the initial clear follicular fluid associated with the existence of an oocyte was used for analysis.The supernatant was separated into aliquots and kept at 20°C before being analyzed.Analysis of serum and follicular fluid parameters According to the manufacturer's instructions, this kit uses an enzymelinked immune sorbent assay (ELISA) based on the Biotin double antibody sandwich technique to measure placental growth factor and sFlt-1.

78(Figure 1 ):(Figure 7 ): 2 ([ 8 ]
Comparison of PlGF levels between PCOS & non-PCOS women (Figure 2): Comparison of sFlt levels between PCOS & non-PCOS women http://doi.org/10.28969/IJEIR.v12.i1.r6.22 et al., Aliyah 79 (Figure 3): ROC curve of serum & follicular fluid PlGF (Figure 4): ROC curve of serum & follicular fluid sFlt (Figure 5): Comparison of fertilization rate between PCOS & Non-PCOS women (Figure 6): Correlations between follicular fluid PlGF & AMH /doi.org/10.28969/IJEIR.v12.i1.r6.22 et al., Aliyah 81 Comparison of ICSI characteristics between PCOS & Non-PCOS women 4. Discussion: In the current study, there has been significantly higher serum PIGF in women who have OHSS as compared to women who do not have OHSS (p<0.001) with cut-off value ≥ 105.3 ng/l was obtained with 72.2 % sensitivity and 85.7 %specificity as well as 79% accuracy.While no studies have documented connections between PlGF and OHSS in the past but, a previous study demonstrated that during ovarian stimulation, vascular endothelial growth factor (VEGF) and VEGF receptor 2 (VEGFR-2) mRNA expression grows and that with the delivery of hCG, each expression reaches its peak (Soares et al., 2008).An in vitro study revealed VEGF-A to be the major motivation of enhanced vascular permeability in OHSS.When VEGF-A binds to VEGFR 1 and VEGF-R1 and VEGF-R2), the receptors are phosphorylated to activate them and transmit the signal to other cell signaling pathways.On the other hand, it has been found that the /doi.org/10.28969/IJEIR.v12.i1.r6.22 et al., Aliyah 82 PlGF binds to VEGFR-1 and its soluble variant sFLT-1(antiangiogenic) but not VEGFR-2 (Albonici et al., 2019 [10]).) from all these give a suggestion that PLGF enhances VEGF-induced vascular permeability causing OHSS in PCOS women .Follicular fluid PlGF has been significantly higher in women with OHSS patients as compared to women with no OHSS (p<0.001) with a cut-off value of follicular fluid PlGF was ≥ 112.2 (ng/l) with 94.4 %, sensitivity, and 85.7 %, specificity as well as 90 % accuracy.Granulosa cells and theca cells both expressed PlGF and VEGFA.Prior to hCG, PlGF and VEGFA levels in follicular fluid were at their lowest.After 36 hours of hCG injection, plGF levels increased sevenfold to their highest levels.(Bender et al., 2018 [6]).On the other side, other study found follicular fluid VEGF, ANG II, and serum VEGF levels were significantly higher in OHSS.Pan et al.From all these data give a suggestion that the PlGF may have a role in the progression of OHSS and its usefulness as a predictor of early and/or late OHSS .Additionally, the Follicular fluids PlGF/sFlt ratio is significantly higher in women with OHSS patients as compared to women with no OHSS.http://doi.org/10.28969/IJEIR.v12.i1.r6.22 et al., Aliyah 83 VEGF-A for binding to sFlt (soluble form of VEGFR-1) so that more VEGF-A is available to activate VEGFR2; thus, by interacting with VEGFR-2, VEGF stimulates new blood vessel development and vascular hyper-permeability (Ceci et al., 2020 [7], Alzaidi, Z et al.,2021[12]).Based on these data, PlGF/sFlt increases as a result of an increase in the PlGF level.

Corbett et al., 2014 [9]). High
when their AMH values are >3.4 ng/ml, AFC is >24, and estradiol levels are >3,500 pg/ml.Ovarian enlargement, release of vasoactive chemicals, ascites, and hypovolemia brought on by an abrupt extravasation of fluid into the interstitial space are the primary events in the pathophysiology of OHSS.(Namavar Jahromi et al., 2018 [4]).OHSS can be classified as early or late depending on when it first manifests, which can predict the prognosis.The symptoms starting nine days after the hCG trigger dosage are due to the exaggerated ovarian response and the exogenously administered hCG's precipitating influence on the ultimate http://doi.org/10.28969/IJEIR.v12.i1.r6.