Endometrial thickness and frozen thaw embryo transfer: A prospective study

Results: The mean age and standard deviation of study population was is 33.26 + 5.95. Out of 69 patients there was clinical pregnancy in 33 patients, miscarriage in 10 patients and ongoing pregnacy in 23 patients. So the implantation rate was 47.83% (33/69), early miscarriage rate (pregnancy ending before 12 weeks) was 14.49%(10/69) and the ongoing pregnancy rate was 33.33% (23/69). Endometrial thickness < 6 mm had comparable implantation rates but low ongoing pregnancy rates.


Introduction
Endometrial Window of Implantation (WOI) is open in the midsecretory phase wherein the markers of invasion and cell adhesion are manifested [1]. The human endometrium has a functional layer and basal layer each consisting of stroma and epitheium [2]. In the functional layer, the epithelium is involved in embryo adhesion and stroma is involved in embryo nurturing [3]. The basal layer epithelium and stroma mainly helps in regeneration [3]. Though several advances have been made in genomics, transcriptomics and the various other interactomes like proteomics and lipidomics of endometrial receptivity, the biophysical markers of endometrial thickness and vascularity are economic and feasible in clinical settings. The classical Noye's histological grading using the LH peak takes into account the seven characteristic histological attributes of mid secretory endometrium that predict its implant ability [3].
The definition of receptive endometrium that is after 7 days of LH surge and after 5 days of progesterone includes eight histological featues. Epithelial features are the fraction glandular cells in mitosis, the visualization of nuclear pseudostrati ication, the presence of secreting basal vacuoles. The stromal features are edema, pseudodecidualization, stromal mitosis and leucocyte in iltration. The assessment of receptive endometrium by ultrasound needs to be assessed as compared to this gold standard of window of implantation [4].
Endometrial Receptivity is integral to implantation so identi ication of an accurate marker of implantation would be highly bene icial in assisted reproductive technology (ART). Despite enormous research in the ield of human embryo implantation, the ideal marker of Endometrial Receptivity remains unidenti ied [5][6][7].
On ultrasound monitering in IVF cycle the endometrial thickness increases in the follicular phase and endometrial character changes from a hypoechoic trilaminar one to a compact hyperechoic look post ovulation [8]. A tri laminar endometrium on the day of ovulation trigger is associated with an increased probability of pregnancy while a hyperechoic character signals failure [9].
In proliferative endometrium there is cell proliferation and cell differentiation and vascularization in response to estrogen. The secretory phase can be classi ied as early secretory, midseretory and late secretory [10]. The early secretory endometrium shows increased intracellular transport and cell metabolism. The midsecretory endometrium or the "Window of implantation" shows immune system signaling and intracellular communication and cell adhesion [11,12]. The late secretory endometrium is mostly in lammatory mediators and apoptosis. The irst half of the sectretory phase is characterized by expression of soluble ligands and their recetors coded by progesterone related genes. The second half of the secretory phase is characterized by vasoconstriction and in lammatory response. Decidual reaction of endometrium happens soon after the closure of window of implantation and this change does not depend on the presence or absence of blastocyst. The activation of innate and humaral immunity characteristic of decasualization are interpreted as 5 line pattern and zonal vascularisation of endometrium [13][14][15]. This study was done to ind the role of endometrial patterns and thickness in predicting and optimizing ART outcome in Frozen Embryo transfer .

Material and methods
This prospective study examined 69 infertile women who underwent embryo replacement from Jan 2019 to Nov 2019 at the ARC Fertility centre, Saveetha Medical College and Hospital. Ethical committee approval and institutional review board approval was obtained (SMC/IEC/2019/11/008). Inclusion criterium was Age group 25-45, two or more blastocyst (grade 3/4) available for replacement and normal uterine cavity in hysteroscopy. Exclusion criteria was presence of endometrial polyp, congenital uterine Anamolies, adenomyosis, endometriosis, hydrosalphinx and ibroid uterus.Written informed consent was obtained from all participants in local language.
The endometrial thickness and pattern was visulised on the day 18 of previous cycle before Frozen Embryo Transfer cycle (Mock cycle). Endometrium was prepared with estrogen started from day 1 of cycle (2 mg/day from cycle days 1 to 4, 4 mg/day from days 5 to 8, and 6 mg/day from days 9 to 12). Progesterone was added after endometrium reched a thickness of 8 mm to initiate the secretory changes (40 mg intramuscular administration of progesterone was provided and maintained for the following 5 days).
Low dose Aspirin (75 mg once daily) was administrated for improving endometrial receptivity. Day 3 Embryos were thawed and embryo replacement was done on day 4 after 3 days of progesterone administration.
From day 8 of cycle 150 IU of HCG was given intramuscularly to promote endometrial thickness reaches at least 7 mm. HCG has been shown to associated with decasualization process signaling via Vascular Endothelial Growth Factor (VEGF), Leukemia Inhibiting Factor (LIF), Insulin like growth factor like Binding protein 1. It also signals the secretion of Matrix mettaloproteinases from NK cells and secretion of Monocyte Colony Stimulating Factor. HCG also inhibits apoptosis promotes differentiation of stromal cells.
Descriptive and inferential statistics were used to analyse the data. Odd's ratio was used to analyse categorical variables. Predictive values of Endometrium > 6 mm was calculated for Biochemical pregnancy, Clinical pregnancy and ongoing pregnancy. Statistical Analysis was done using MEDCALC (Belgium).

Results and analysis
A total of 69 patients with history of thin endometrium who underwent frozen ET after treatment in ARC Fertility centre, Saveetha Medical College over a period for one year were analyzed in this study. The mean age and standard deviation of study population was is 33.26 + 5.95. Out of 69 patients there was clinical pregnancy in 33 patients , miscarriage in 10 patients and ongoing pregnacy in 23 patients. So the implantation rate was 47.83% (33/69), early miscarriage rate (pregnancy ending before 12 weeks) was 14.49%(10/69) and the ongoing pregnancy rate was 33.33% (23/69). Table 1a shows the clinical pregnancy (fetal pole and cardiac activity) diagnosed by ultrasound in group 1 and group 2. There were no signi icant differences in implantation rates in thin and thick endometrium. Table 1b shows the ongoing pregnancy (till second trimester) diagnosed by ultrasound in group 1 and group 2. Table 1b shows that there are fewer ongoing pregnancy in thin endometrium as compared to thick endometrium.This implies that though there are implantations in thin endometrium there are more chances of miscarriage and there is less chance of ongoing pregnancy if endometrium is less than 6 mm.   Table 2 tells us the sensitivity, (speci icity, likelyhood ratio positive and negetive and positive and negetive prognostic value of Endometrium thickness > 6 mm in predicting the ongoing pregnancy rates (Table 2).

Discussion
Endometrial thickness assessment is done routinely before embryo transfer and thin endometrium is considered as poor predictor and embryo transfer is deferred. However one must remember that there are isolated case reports of successful implantation in very thin endometriums also [16][17][18]. In 2 D ultrasound the endometrium is classi ied as Grade A, B and C. The Grade A endometrium the 5 layered endometrium and the intervenning area between the outer and inner echogenic areas is more echogenic than the surrounding endometrium. In Grade 2 endometrium the intervening is less echogenic than the surrounding endpmetrium. In Grade 3 endometrium the enometrium is homogenously isoechoic. This changes successively represent the rise and release of glycogen containing vacuoles up to the endometrial surface [19].
On Colour Doppler the vascularity can reach to varius zones as characterised by Applebaum's [20]. The outermost vascularity zone is subzonal vascularity. Blood vessels reaching the 2 mm thick area surrounding the the hyperechoic outer layer of the endometrium. Zone 1 is the outermost myometrial endometrial junction. Zone 2 is the hyperechoic outer endometrium. Zone 3 is the inner hypoechoic endometrium. Zone 4 is the endometrial cavity. The reasons for low implantation could be a high impedance blood low of the radial arteries leading to poor endometrial glandular growth in thin endometrium. Poor angiogenesis subsequent to decreased VEGF secretion can also affect endometrial vascularity. This in turn increases chances of poor placentation and inappropriate vascularization leading to early abortions even if pregnancy is established. Transfer of embryos to an endometrium prepared by HRT seems to yield better results than fresh embryo transfers [21][22][23]. The other parameters included assessing uterine biophysical pro ile for implantation are summerised in table 3.
Other agents that are used to improve endometrial thickness and vascularity are Filgrastin Intrauterine instillation, Vitamin E, L arginine and silden il citrate [24][25][26].

Conclusion
Since Noyes in 1950's the endometrium receptivity has been a challenge. Transcriptomics and DNA probes are not full proof as they have probes which carry inbuilt problems of speci icity and sensitivity . Decidualizing endometrium can be easily pro iled by sonoendocrinology thereby avoiding expensive tests of biochemical hormones and transcriptomics.