Evaluation of serum VEGF-A and interleukin 6 as predictors of angiogenesis during peri-implantation period assessed by Transvaginal Doppler Ultrasonography amongst women of prior reproductive failure: a cross-sectional analytical study.

1. Non pregnant women of age group between 20-40 years.

2. Primary or secondary infertility patients having unexplained infertility with previous stimulated or unstimulated cycles.

3. Patients with history of one or more abortions.

4. Willing to give consent.

1. Patients not giving consent.

2. Patients with anatomical (uterine, tubal or cervical abnormalities diagnosed with ultrasonography or hysterosalpingography).

3. Patients with chromosomal abnormalities diagnosed by chromosomal study will be excluded.

4. Patients with autoimmune or endocrinological disorders diagnosed by blood tests or clinical features.

5. Patients with chronic illness or infective aetiology which could be a cause of infertility or abortion.

1. A Study conducted in the Department of Immunology and Allergy, St Helier Hospital, U. K by Rhea Bansal, Brian Ford et al., titled Elevated Levels of Serum Vascular Endothelial Growth Factor-A Are Not Related to NK Cell Parameters in Recurrent IVF Failure. Background of the study was to establish relation between VEGF and NK cells in recurrent IVF failure patients as both the factors are responsible to play an important role in vasculogenesis and angiogenesis.

It was a case control study conducted in 62 women with repeated reproductive failure which was failed 3 attempts of IVF with fresh eggs, with 72 normal fertile women as control group. Women with autoimmune, endocrinological, anatomical factors associated with reproductive failure were excluded. Samples were collected in proliferative phase of menses and those women who underwent hormonal therapies were called for sample atleast after 2 months. The levels of VEGF-A, its receptor VEGF-R1, were estimated by ELISA kit and NK cells level by flow cytometry as anti-CD56, anti-CD16, anti-CD3 and anti- CD69 and NK cytotoxicity were determined.

Result: The serum VEGF-A levels measured in 62 women with recurrent reproductive failure was significantly raised compared to the 72 healthy controls [median 362.9 pg/ml vs. 171.6 pg/ml). The serum soluble VEGF-R1 levels in the RIF group were similar to that of the control group. The median total NK count was 9.25%, the median NK CD69 count was 0.555×106/L and finally the median NK CD69 was 0.14×106/L. There was no correlation between these results and the corresponding VEGF-A and VEGF-R1 levels. The median killing of NK cells was 27.5% at 50:1, 19.5% at 25:1 and 12% at 12.5:1. There was no correlation between the NK cytotoxicity values at each effector: target ratio and serum VEGF-a and their receptor.

Conclusion of the study was that plasma VEGF levels were significantly raised in women with recurrent reproductive failure as compared to fertile women. No relation between VEGF levels and NK cells were found.²⁶

2. In a study conducted in the USA in the year 2014, in Obstetrics and Gynaecology department at Mercer School of Medicine, Central Georgia Fertility Institute, USA by Abdelmoneim Younis et al., the values of Interleukin 6, serum TNF-α, MCP-1 and paraoxonase-1 levels of women with recurrent pregnancy failure, endometriosis and PCOS were studied and evaluated.

Method: The study included thirty-six patients with Unexplained infertility, Endometriosis, PCOS who underwent controlled ovarian stimulation for either In vitro fertilisation or Intra uterine insemination. After their informed consent, serum sample were collected first on day 3 and again towards the end of treatment with Follicle Stimulating Hormone during (peak) and evaluated for the values of Interleukin-6, Tumour necrosis-α, MCP-1 and Paraoxonase-1.

The study group included thirty-six women with infertility (average of 33.7 ± 4.7 years within range of 26–44 years). The infertility women were further divided into three groups of infertility as: 1. Recurrent pregnancy failures (19.4%), 2. PCOS (38.9%) and 3. endometriosis (41.7%). All the women had normal puberty onset, sexual development and normal blood investigations. The patients were selected on the basis of normal blood investigations, usg pelvis, hormonal studies and husband’s semen analysis. Unexplained infertility patients had all the normal profiles of both partners. Patients with cause of infertility as endometriosis underwent laparoscopy for classification. All the patients were given recombinant human follicle-stimulating hormone (rFSH) for controlled stimulation of ovaries. Injection was given once a day for 7–8 serial days and the development of follicules were monitored serially using serum E2 levels and TVS. An hCG injection was given 34 to 36 hours before the rupture of follicle to trigger maturation and rupture of dominant follicle. Patient was subjected to IVF or ICSI according to semen quality. Fertilized embryo was assessed for the cleavage and the embryos were transferred between day 3 and 5. Pregnancy was confirmed on day 14 of insemination using a positive serum β-hCG (>25 mIU/mL) or by transvaginal ultrasonography done at around 7 weeks after insemination to see the gestational sac for foetal pole and cardiac activity.

Results obtained showed that the levels of IL6, MCP 1 and Paraoxonase-1 peaked positively along with peak of Estradiol levels. Mean of Tumour necrosis factor-α levels were statistically significant in Unexplained infertility group (P < 0.05). The serum levels of IL-6, and MCP-1 were significantly (P < 0.05) more in women with PCOS in contrast with that of Endometriosis and Unexplained infertility group. Tumour necrosis factor-α levels were negatively related to oestradiol levels and was less in pregnant women as compared to non-pregnant women (P < 0.05). There was no significant difference amongst the three groups when compared for patient’s age, FSH day 3 level, BMI, PON-1 and pregnancy outcome.

Conclusion generated out of the study was that the ovarian stimulation was the main factor for increased levels of circulating cytokines as seen by raised levels of PON-1, MCP-1 and IL-6 and reduced level of TNF-α after controlled stimulation of ovaries. Evidence of connection between mild endometriosis and cytokines were not established, PCOS was drawn to have relation with raised serum MCP-1 and IL-6 levels and lesser TNF-α level. No relationship could be found between PCOS and PON-1 level, mild endometriosis or recurrent pregnancy failure. Unexplained infertility was seen to have elevated TNF-α level.²⁷

3. Another study conducted in the Department of Obstetrics and Gynaecology, Uludag University School of Medicine, Turkey in 2016 by M. A. Atalay et al., showed clinical correlation of maternal serum vascular endothelial growth factor (VEGF) levels with idiopathic recurrent miscarriages.

In this study, 84 patients were sorted out in recurrent miscarriages group who had three or more spontaneous abortions and 47 women were identified with actual spontaneous miscarriages of which 25 women with idiopathic miscarriage were selected based on normal karyotyping, no infection, no autoimmune or hormonal imbalances. 25 women were followed up for study out of which 21 women conceived spontaneously who constituted the case group. They were compared with the control group which had 24 normally conceived women. Transvaginal obstetric ultra-sonographies were done and maternal sample collection weas done between 5^th and 10^th gestational weeks to compare serum VEGF and progesterone (P 4) levels. ELISA technique was used to measure VEGF and P4 levels.

Results showed that the idiopathic cases amongst all the recurrent abortions were with 44.7% prevalance. Serum VEGF levels had no significant change when seen in the gestational age in the recurrent abortion and the control groups (P = 0.72 and P = 0.89, respectively). There was positive result between serum VEGF levels and the patients’ age in recurrent abortions group (r = 0.515). Median of serum VEGF value was found higher in recurrent abortions group when compared to control group which was significant (210.33 ± 108.23 pg/ml vs.123.91 ± 18.8 pg/ml, respectively). There was no difference found in the idiopathic recurrent abortion group and control group in median P4 levels (19.53 ± 5.79 ng/ml and 20.08 ± 7.85 ng/m l, respectively). All different possibilities of recurrent abortions amongst patients (n = 22) were uterine anomalies (n = 5, 23%), autoimmune causes (n = 4, 18%), endocrine disorders (n = 4,18 %), hereditary thrombophilia (n = 7, 32%) and parental balanced translocations (n = 2, 9%). Prevalence of the idiopathic causes (n = 25) was 53%.

Conclusions were as follows: A positive correlation between VEGF levels and recurrent abortions were found. Serum VEGF level had no relation with gestational age. Serum VEGF levels were positively correlated with maternal age. Increased maternal age above 35 years have positive relation with serum VEGF levels.²⁸

4. A study was conducted to see the essence of various cytokines on reproductive system conducted in the department of microbiology by Batool Mutar Mahdi et al. in the Al-Kindy College of Medicine, Baghdad, Iraq, on women with reproductive failure to assess serum levels of anti-inflammatory cytokines like (IL-10 and IL-6) with pro-inflammatory cytokines like tumour necrosis factor (TNF-a) and interferon-gamma (IFN-c).

Methodology: study design was cross- sectional, workup was done at a Hospital in Bagdad, Iraq from 2008 to 2010. 45 women with unexplained infertility were taken up for the research purpose. The study group consisted of women with normal ultrasonography, hysterosalpingography, husband’s semen analysis. Other factors of infertility like tubal, ovarian, cervical, uterine, autoimmune, peritoneal and hormonal were excluded. Serum levels of cytokines (IL-10, IL-6, IFN-gamma and TNF-alpha) were measured using ELISA and comparison with control group consisting of ethnically matched 30 fertile women were done.

Results were as follows: Cytokine profile showed that there was a statistical increase in IL-10 (18.09) (P = 0.002) and IFN-c (49.62) (P = 0.0001) in patients of unexplained infertility. IL-6 and TNF-alpha showed no significant difference in fertile patients. There was no correlation in the average age of infertile women group - 32.2 ± 6.1SD (range- 20-40 years) with average age of fertile group which was 30.4 ± 9.1 SD (range - 19-39 years). There was also no correlation in BMI.

Conclusion was that there was significant increase in IL-10 and IFN-c in women with unexplained infertility.²⁹

5. Another study was conducted to see the correlation of pro – angiogenic factors (VEGF-A and C levels) and clinical features of women having repeated pregnancy failure and compare with healthy pregnant females of same age group and characteristics features. It was a case control study conducted in Kasturba Medical College, Manipal by Bagheri et al. in the Department of Biochemistry which included 90 non- ANC women with previous history of recurrent pregnancy losses with age-matched 2 control groups as follows: 1. 70 non-ANC women with no previous history of repeated miscarriage with minimum single live child (controls) and 2. 70 ANC mothers without previous history of repeated abortions with a minimum one live child (controls). Those with undetermined recurrent abortions were included in the study in which other factors of recurrent abortions like endocrinological, anatomical, immunological and infectious factors were ruled out. Demographic and anthropometric data were collected via preset questionnaire and serum levels of VEGF-A and C were measured by ELISA kit.

Result showed that levels of VEGF-A and C were significantly less in recurrent spontaneous abortions group (189.87 ± 88.1 vs 238.8 ± 99.6) when compared with healthy non pregnant first group (239.1 ± 99.7 vs 275.5 ± 133.08) and pregnant second group (301.5 ± 76.4 vs 402.5 ± 128.6). it was also observed that the clinical characteristic factors were significantly associated with concentration of VEGF A and C levels in cases and controls. The result also showed negative correlation of serum VEGF A and C levels when compared with cases and controls in age and BMI.³⁰

6. In a study conducted to Evaluate serum levels of Pro-inflammatory Cytokines in cases of Idiopathic Repeated Pregnancy Losses by Poonam Tyagi et al., Department of Medical Laboratory Sciences, College of Applied Medical Sciences, Prince Sattam Bin Abdulaziz University, Al-Kharj, Riyadh, Saudi Arabia, in the year 2020, a total of 100 patients of age group 21-41 years were taken up for study and were divided into two group: Fifty pregnant females with history of idiopathic recurrent abortions were taken under study group and 50 healthy pregnant women were recruited under control group. Idiopathic recurrent abortion was excluded by ruling out of autoimmune, anatomic, infectious, genetic and endocrinological causes. Control group had fifty normal healthy pregnant women who attended outpatient department of obstetrics and gynaecology for a routine check-up, having <20 weeks of gestation with previous one live birth, and without any history of the treatment for miscarriage or infertility with normal ultrasonography. Blood sample were collected for estimation of cytokines as interleukin (IL)-6, TNF – alpha, IL-18, IL-12, IL-8 and interferon (IFN)-γ using ELISA kit.

There was no relation found between all the demographic characteristics except number of abortions in case of study group patients (P ˂ 0.001).

FSH level was statistically significant (6.14 ± 1.32) in cases of women with recurrent miscarriage group in comparison to pregnant women of control group (9.13 ± 1.10) (P ˂ 0.001).

The mean with standard deviation of the level of IL-6 cytokine level was found lower (79.63 ± 1.49) in women with recurrent abortion group in comparison to pregnant control group females (93.26 ± 1.63) with P value ˂0.001. The increased level of IL-8 cytokines (72.97 ± 1.67 pg/ml) was observed in pregnant females with previous history of idiopathic recurrent abortion as compared with normal healthy pregnant controls (64.67 ± 1.36) (P ˂ 0.001). The high levels of IL-12 cytokines (51.51 ± 1.97) found in patients with previous history of abortion group was identified as compared to healthy pregnant controls (18.23 ± 3.13). The result was significant statistically (P ˂ 0.001). The mean ± SD level of IL-18 type of cytokine level was found significantly increased (596.18 ± 99.16 pg/ml) in patients having a history of recurrent abortions in comparison to controls (329 ± 101.18) with (P ˂ 0.001). Mean ± SD level of IFN-γ was also observed elevated significantly (216.86 ± 59.86) in serum of women of idiopathic recurrent abortions as compared with pregnant women (123.12 ± 61.81) (P ˂ 0.001). Increased production of TNF-α (335.13 ± 4.97) was measured in case of study group patients with previous history of recurrent abortions in comparison to normal healthy pregnant women (180.79 ± 4.12) with (P ˂ 0.001).

Results: Th1 activity (IL-18, IL-8, IL-12, TNF-α, and IFN-γ) were found to be more in first group of patients with recurrent abortions irrespective of pregnancy outcome as compared with Group II controls (P < 0.001) while (IL-6) which belongs to Th2 group was found decreased in Group I patients of recurrent abortion (P < 0.001) when compared with normal control Group II.³¹

7. In a study conducted to see the correlation between vascular endothelial growth factor and 3D doppler ultrasonography with sub endometrial wave like movement in case of unexplained infertility by Eman,M.et al., in the Department of Obstetrics and Gynaecology, Faculty of Medicine for Girls, Al-Azhar University, Cairo, Egypt, from September 2016 to September 2019, the Relation between Vascular Endothelial Growth Factor (VEGF),sub endometrial blood flow with doppler by 3D as indicator of uterine receptivity in spontaneous as well as stimulated cycles in unexplained infertile women were evaluated during the implantation window. Total of 96 participants were recruited. They were put into three groups. Group one was: thirty-two patients with unexplained infertility with unstimulated cycle, second group was thirty-two patients of unexplained infertile women with stimulated cycle (clomiphene citrate) and third group was thirty-two participants normal fertile seeking contraception as control group. Groups (A and C), first and third group were asked to come during mid luteal phase for examination while patients in group (B), second group, were asked to come first in follicular phase and they were given clomiphene citrate drug 50 mg twice a day for 5 days starting from day 2 of cycle. Follicles were monitored till 18 mm of size and then they were given HCG and cyclogest rectal suppository for luteal support and were called on day 21-22 to see for rupture.

All participants were called on day 21-22 day of cycle for 3D transvaginal ultrasound to detect sub endometrial blood flow, sub endometrial wave like movement and on the same day venous blood sample was also taken to measure serum VEGF.

Result was found as follows: There was a statistically significant decrease in below endometrial blood flow among infertile groups (A and B) than fertile group (C) with P value <0.001. Serum levels of VEGF during peri implantation period was found to be lower in group A and B infertility group when compared to group C of fertile controls. There was statistical significance between VEGF levels and Doppler blood flow study to sub endometrial region with P value 0.023. There was no relation found in all 3 groups regarding demographic features. It was found that the serum concentration VEGF levels of <200 pg/ml was able to predict good endometrial receptivity with a sensitivity of 100% and a specificity of 96.8%.

Conclusion out of the study was that transvaginal colour Doppler study of the sub endometrial blood flow distribution is an effective method to evaluate endometrial receptivity. The junctional zone is to be considered as a separate functional unit within the uterine cavity and plays a significant role in the processes of implantation. Serum VEGF levels were found to rise with increasing Doppler vascular penetration zones which indicates that serum VEGF levels is a good marker to determine endometrial receptivity.³²

8. The study comparing serum levels of VEGF in normal early pregnant women with women of recurrent pregnancy loss was conducted in the Department of Anatomy, Lady Hardinge Medical College, New Delhi, India by Dr. Renu Baliyan et al., which was a descriptive comparative study. Total 120 pregnant patients were recruited for the study, divided in two groups: a study group (60 women) and control group (60 women). The points on which they were taken for the study group were age group between 20-35 years with 3 or more consecutive recurrent pregnancy losses. Subjects had monogamous relation with no anatomical, infective, autoimmune, hormonal, or chromosomal factors which could cause recurrent pregnancy loss. The women in control group were of same age group without any history of abortions, intra uterine demise or still births and at least one live birth. After detailed history taking and general examination with systemic examinations, the blood sample was drawn from the women for VEGF levels. The correlation of serum Vascular Endothelial Growth Factor (VEGF) with recurrent miscarriage was studied.

RESULTS: The variation was statistically significant with serum levels of VEGF found to be lower in recurrent abortion group as compared to normal control group. 50% of women in study group had levels below 100 pg/ml and another 48.3% had levels between 100-200 pg/ml as compared to controls who had VEGF levels between 1000-2000 pg/ml in 65% and between 2000-3000 pg/ml in another 20%.³³

9. Another study dates back to year 1997 which was conducted to know the levels of VEGF in the serum as well as in the follicular fluid of patients who underwent IVF by Annette Lee et al., at the Oregon Health Science University, Portland and Oregon Regional Primate Research Centre, Beaverton, Oregon, U.S.A. The study was conducted to assess the relationship of VEGF in the serum and follicular fluid (FF), Estradiol levels, and Progesterone levels in patients subjected to IVF; then to assess in early pregnancy the relationship between beta Hcg and serum VEGF levels; and also to observe and manage a case of severe ovarian hyperstimulation syndrome (OHSS) admitted for observation and management when she was subjected to IVF with her serial measurement of VEGF levels in serum and ascitic fluid. Study design was a prospective observational study. Women who underwent conventional In vitro fertilization (IVF), receiving either donor oocytes or who conceived spontaneously were included in the study. One patient had to be hospitalized with severe OHSS during the course of study period was also included to see her response and management.

This study had three parts. In first part, serum sample and Follicular fluids were obtained from patients who underwent IVF, 34 to 36 hours after ovulation during egg retrieval. In second part, women who conceived after autologous fresh embryo transfer (ET) or those who underwent IVF from the donor eggs or even women who conceived spontaneously after unstimulated cycles were taken for serum samples during the late luteal phase or early pregnancy. In third part, a patient who became pregnant and later landed up in severe OHSS following the IVF cycle done for male factor infertility was admitted, her serum and ascitic fluid samples were taken. A total of 36 patients; age between 25 to 42 years, who underwent ovarian stimulation for IVF according to a standardized pre-set protocol were studied.

For women under part 1 of study, Leuprolide or nafarelin acetate were given during the mid-luteal phase of previous cycle as pituitary down-regulation drug starting from 3 rd day of induced menses. Urinary FSH dose was individualised and given. Serial monitoring of the follicular size by serum E2 levels and transvaginal ultrasonography were done. hCG was administered 7500 IU, when atleast 2 follicles reached 18 mm diameter for follicular rupture. Oocyte collection was done 36 hours later under ultrasonographic guidance. Embryo transfers were done on day 3 after eggs retrieval. In 13 patients, peripheral venous samples were drawn before oocyte retrieval, centrifuged, and the serum stored for evaluation of E2, VEGF and P. Follicular Fluid (FF) from the largest follicle seen in the mid cycle was taken from all the cases and the supernatant were stored for further evaluation of E2, P and VEGF levels. In another 6 patients, FF were separately collected from each ovary based on single largest follicle. So, a total of 42 follicular fluid samples were obtained from 36 procedure of egg retrieval. For luteal phase support, protocols were different. Patients of first group who received autologous fresh embryos were given hCG 2500 IU for 5 days after egg retrieval and 100 mg of micronized progesterone oral dose thrice a day, starting from second day of retrieval till the day of embryo transfer and intravaginal insertion thereafter. Second group women who received donor eggs were given 100 mg of oily preparation of progesterone IM daily starting from two days before Embryo transfer. Luteal phase support was given till they either had a 12 weeks pregnancy or a negative serum hCG.

In case of study group 2, women who became pregnant after autologous fresh ET (n = 8) transfer and women who received embryos from donor oocytes (n = 7), their serum samples were collected. 2 samples of serum were taken at different intervals: the first between 11-14 days and the second between 15-17 days after Embryo transfer. Recipients of donor oocytes were given GnRH agonists for pituitary down-regulation and E2 and P replacement therapy as luteal phase support. All samples of serum were analysed depending on the number of days after Embryo transfer that is (day 11-14 or day 15-17). As measure of control, serum samples from patients who underwent IVF but who did not concieve after autologous fresh ET were taken between (days 11-14; n = 8) and also from women who conceived spontaneously without any cycle of induction (n = 8). In the second group, 2 samples, starting 5 weeks from the first day of the LMP were collected 48 hours apart. It was seen that all the women who became pregnant carried their pregnancies beyond the first trimester.

In study 3, a patient who became pregnant after embryo transfer and later had triplets landed up in severe OHSS because of ovulation induction for IVF, done in view of male factor infertility. Her serum and tapped ascites Fluid samples were collected for measurement of VEGF levels. She was given 30 ampules of urinary FSH in view of ovulation induction and her serum E2 was 2,534 pg/ml on the day of hCG administration; total of 27 eggs retrieval were done. After 3 days, four fresh embryos were transferred. She presented on 7^th day after ET with nausea, pain in abdomen, vomiting and moderate abdominal distention with features suggestive of hyponatraemia, ascites and haemoconcentration few days later. She was hospitalized for severe OHSS management. In view of respiratory discomfort her therapeutic paracentesis was performed twice. Serial serum as well as ascites fluid samples were collected and evaluated for VEGF level estimation. For the purpose of comparison, peritoneal fluid sample were also collected from 4 women between 30-35 years of age who had no pelvic pathology, who had come for elective tubal ligation during laparoscopy.

Result were as follows: in study group 1, Follicular fluid VEGF concentrations of an average of 356 pg/mL in 13 patients were tenfold greater which was significant (P ~ 0.01) than serum VEGF levels with mean of 50 pg/ml at the time of oocyte retrieval. Follicular fluid levels of VEGF and P were positively correlated (P < 0.01). Follicular fluid VEGF levels also had positive correlation with serum P levels (P < 0.01) and also with patient’s age (P < 0.05) but had no correlation between serum or follicular fluid E2 levels neither with the counting of oocyte retrieved. The patients in whom serum VEGF was measured after autologous ET, those patients who later got pregnant had raised (P ~ 0.05) serum VEGF levels on 11-14 days as compared to those who did not concieve or who conceived after donor eggs transfer. However, serum VEGF levels declined significantly (P ~ 0.05) in the pregnant recipients of autologous embryos between 15-17 days. There were no statistical significance in serum VEGF levels between days 11-14 or days 15-17 in pregnant recipients of donor eggs. In case of patient who was admitted for management of OHSS, serum VEGF levels varied from 168 to 292 pg/mL during controlled ovarian stimulation before hCG administration and egg retrieval, and then it increased to a maximum of 1.165 pg/mL with the onset of symptoms (10 days after egg retrieval), before decreasing to 486 pg/mL as symptoms resolved during her hospitalization. The recurrence of symptoms at 21 days after retrieval again showed elevated VEGF levels (733 pg/mL). In contrast, two ascitic fluid samples had VEGF levels of 202 and 137 pg/mL, which were not significantly different from control values (149 + 8.7 pg/mL).

Conclusions drawn out of the study was that patients who underwent IVF, the levels of VEGF in follicular fluid at the time of oocyte retrieval was related positively with the depth of follicular luteinization. There was also a significant role of ovary in the serum VEGF levels during early pregnancy. High levels of serum VEGF can be a predictor in the development of OHSS features.³⁴

10. In another study done in women with recurrent implantation failure, endometriosis and abortions to measure the polymorphism in +405 G/C VEGF A after ICSI-ET Cycles by V Penchev et al., at the Department of Assisted Reproduction, Medical Centre, Bulgaria, in the year 2014-15, the main background behind conducting study was that almost one in six partners who are in reproductive age worldwide experienced infertility. In such cases, various assisted reproductive techniques (ART) can be utilized to obtain a healthy pregnancy and childbirth. But a large percentage of ART attempts fail and do not give pregnancy or even abortion. The successful implantation of embryos are dependent on various factors like trophoblastic and syncytotrophoblastic proliferation, migration of embryo towards uterine cavity, adhesion and invasion into the endometrial layers. These are mediated by various growth factors and hormones produced locally. VEGF was found as an important factor which play a vital part in human vasculogenesis and embryogenesis.

Methodology of the study included study of 41 patients and 23 controls who were subjected to comparison of 3 genotypes which regulated angiogenesis. Study group had 41 patients who were divided according to the subtype of study into 3 subdivisions as patients with recurrent implantation failure, endometriosis and recurrent miscarriages and the control group had 23 participants who were naturally conceived pregnant women. Peripheral blood sample was collected for DNA typing. Genomic DNA was then recruited from blood samples using standard protocols. PCR reaction forwarded by Restriction Fragment Length Polymorphism assay (PCR-RFLP) by BsmFI restrictase were performed to obtain VEGF +405 G/C allele. RTPCR genotyping with 5’ exonuclease technology and Sanger capillary sequencing were used as gold standards to confirm the result. The method used for ovarian stimulation, embryo cultivation and embryo transfer along with medications given for luteal phase support were same in all the cases.

Results: The C allele frequency was found to have statistical significance in patient’s group as compared to control group (53.6% vs 24%, OR = 3.68, 95% CI, P < 0.002). There was also statistical significance in the incidence of the +405 C/C genotypes in women of patient group as compared to control group (37% vs 4%, OR = 16.2, 95% CI, P < 0.02).

Conclusion of the study was as follows: There was positive correlation between the VEGF A +405 C/C genotype and the factor for development of endometriosis and spontaneous abortions. In the recurrent implantation failure group, the results did not show any such correlations. The receptor polymorphism gene testing can be advised to patients with indications and can be properly followed up and managed for infertility. The study group needs to be expanded and it should include more genetic polymorphism so as to get more association with recurrent pregnancy losses, implantation failures and endometriosis.³⁵

11. In yet another study conducted by Dr. Priyanka Banerjee et al., the main factors causing vascular dysfunction in idiopathic recurrent miscarriages was conducted at Kolkata, India in the Institute of Reproductive Medicine and in the Indian Institute of Technology, Kharagpur in the year 2013. Total 66 patients were recruited for the study in age less than 35 years and Body Mass Index less than 28, with previous history of 3 or more recurrent abortions within the first 12 weeks of gestation with no pathological cause. They were done with a series of investigations to rule of probable anatomical, chromosomal, autoimmune, endocrinological, genetic and infectious causes. Any women found having any abnormal laboratory or clinical parameters were excluded from study. They were compared with 50 women of same age group who were undergoing sterilisation and had no previous history of abortions or mishaps in pregnancy as control group. The patients were followed up with serial ultrasonography (USG) to measure follicle growth from day 10 onwards till ovulation. Endometrial biopsies were obtained from all patients of both the groups in between 18^th and 22 nd day of their menses after confirming ovulation under GA by D & C and sample obtained were sent for histopathological evaluation and estimation of various cytokines and growth factors in endometrium.

Results were as follows: No statistical significance were seen in demographic features, BMI, serum oestrogen levels, endometrial thickness and progesterone levels in patients of study group and control group. USG doppler done of the endometrial layers showed that the S/D ratio, PI, EDV were significant in women of study group as compared to control group. However, RI was found to be comparable between the two groups.

Levels of various vasodilators like eNOS, ADM and NO which were measured during implantation window were found to be less in study group patients as compared to control group. IL-1β, TNF-α, IFN-γ, TGF-β1 and PGE2 were found to be higher and IL-4, IL-10, IL-2, 6, 8 and VEGF were lower in study cases as compared with control cases. PECAM-1, which is a marker of vasculogenesis and angiogenesis, showed lower immunoreactivity in patients of study group as compared to strong immunoreactivity in control group. A significant positive correlation were obtained between IL-10 and PI (r = 0.79, P < 0.001), VEGF with EDV (r = 0.61, P < 0.001), and eNOS with EDV (r = 0.74, P < 0.001). The S/D ratio also showed a significant negative correlation between VEGF (r = -0.78, P < 0.001) and eNOS (r = -0.67, P < 0.001), respectively.³⁶

12. Another study conducted titled: Aberrant cytokine production from peripheral blood mononuclear cells in recurrent pregnancy loss by M.D.Bates, S. Quenby, K. Takakuwa, P.M.Johnson and G.S.Vince in the Department of Immunology and Department of Obstetrics and Gynaecology, University of Liverpool and Department of Obstetrics and Gynaecology, Niigata University School of Medicine, Japan.

BACKGROUND of the study was: Successful pregnancy was dependent on an increase in Th2-type cytokine response, while a poor pregnancy outcome was associated with lower Th2 cytokines and an increase in Th1 cytokines. This prospective study was conducted to see the correlation between recurrent pregnancy loss and the cytokine response.

METHODS: 46 women with three or more recurrent miscarriages were recruited into the study and when they missed periods or confirm pregnancy were followed up if ultrasonography had a gestational sac with either a yolk sac or a foetus with cardiac activity between 6 to 10 weeks of gestation. Patients were followed up with ultrasonography for reassurance until 12 weeks gestation and later they received routine antenatal care. Patients with antiphospholipid antibody syndrome, endocrinological causes like (oligo-menorrhoea, abnormal thyroid function tests), chromosomal causes (maternal or paternal balanced translocation) or uterine structural abnormalities (assessed by cervical weakness only) were excluded from the study. One patient was also evaluated at eight weeks of gestation when a foetal cardiac activity was detected and two weeks late demise was diagnosed. Total 25 healthy pregnant women who underwent elective termination of pregnancy at 6-10 weeks of gestation were taken as gestationally age-matched controls. These women had no previous abortions. 11 non-pregnant women were also taken for blood sample collection at random points in the menstrual cycle.

RESULTS: Production of IFN-γ was found to be low in pregnant women than in non-pregnant women and even lower in women with recurrent abortions (P0.0191). IL-10 was increased in pregnant women as compared to non-pregnant women, and increased in RPL women (P0.026). IL-4 was also increased in women with RPL (P0.0001). No differences in IFN-γ, IL-10 or IL-4 levels were obtained in RPL women who aborted later with those who gave live birth. RPL women who later became pregnant had similar concentrations of TNF-α to pregnant women, RPL women who later aborted had significantly lower levels of TNF than either pregnant women (P0.02) or non-pregnant controls (P0.0004).

CONCLUSIONS: The cytokine shift, which characterizes normal pregnancy, was increased rather than decreased in RPL pregnant women.³⁷