Endometrial Heparin-binding Epidermal Growth Factor Expression

Women with PCOS comprise a majority of fertility clinic attendees. Unfortunately, a high failure rate following fertility treatment was observed especially in obese women due to implantation failure. The local study on PCOS women has shown significant changes in an endometrial tumor - regulatory genes but not focusing on the endometrial implantation failure. Many previous attempts using human chorionic gonadotrophin (HCG) infused embryo, gonadotrophin agonist therapy or progesterone support aiming to improve implantation failure in the assisted reproductive technique still unable to enhance pregnancy rate beyond 40% despite a higher' fertilization rate up to 95%. There is still a research gap on what makes obese PCOS women prone to coincides with implantation failure. Endometrial component related to the expression of growth factors play an integral role in establishing cellular context necessary for successful pregnancy. Thus, a new fundamental knowledge on endometrial specific heparin-binding epidermal growth factor expression in the obese PCOS women is vitally important, not only to predict implantation failure but a potential therapy to improve pregnancy outcome.

This prospective study is going to be performed at UKM Medical Centre for a duration of 1 year. The PCOS and control women will be recruited from the Medically Assisted clinic in Obstetrics \& Gynaecology department.

The sample size of this study is calculated using Power and Sample Size Calculator by Dupont \& Plummer 1998 for paired t-test response version 3.1.2; using the endpoint of mean of Hb-EGF expression during window of implantation in predicting successful pregnancy following in vitro fertilization (IVF) rate by Mengling et al. 2016 (11) as one of its specific outcome. The auto-generated sample size using this programme is 8 subjects. Considering the dropout rate of the sample is 30%, the total sample size required in this study is 10 subjects each arm, making a total sample size 40.

The participants are divided into four groups according to PCOS diagnosis and Asian adult population body mass index (BMI):

1. Anovulatory PCOS women with a BMI greater than 27 (OB-PCOS)
2. Anovulatory PCOS women with a BMI lower than 25 (NW-PCOS)
3. Healthy fertile women with a BMI greater than 27 (OB-C)
4. Healthy fertile women with a BMI lower than 25 (NW-C)

In the PCOS group, generally anovulatory cycle hence the implantation window during mid-secretory endometrium can be exhibit following a daily oral micronized progesterone (Utrogestan 200mg) for 10 days based on previously published methods.

Volunteers in the control group with a normal regular menses will be counseled regarding the procedure and monitored for ovulation. The endometrial biopsy will be acquired during implantation window (mid-secretory endometrium), which occurred 7-9 days after the ultrasound and urinary LH confirmed ovulation.

Endometrial samples are obtained using the Pipelle de Cornier catheter for all participants. The endometrium sample that is taken is divided into two portions; a portion for histopathological examination (HPE) for endometrial dating and a portion frozen in liquid nitrogen at -80 degrees Celsius for real-time PCR analysis.

For endometrial dating, all samples were analyzed by classical histological analysis according to the criteria of Noyes.

A total amount of RNA was isolated from the endometrial tissue using RNA easy kit by following manufacturer's instruction. Pellet will be suspended in 30ul of RNAase-DNAse free water. Finally, nanodrop will be used to determine the concentration and quality of RNA. By following manufacture's instruction, total RNA will be reversed transcribed to cDNA. The target for cDNA is primers for heparin binding epidermal growth factor. QRT-PCR will be performed using Sybergreen Master Mix and detected using QRT-PCR detector machine in accordance with manufacturer's protocols. The reaction was running in real-time PCR machine with 40 cycles and cycling temperature as follows: 95 ºC for 10 minutes and 15 seconds, 56 ºC for 30 seconds and final dissociation stage: 94 ºC for 15 seconds, 56 ºC for 15 seconds and last 95 ºC for 15 seconds. Relative quantification will be calculated by normalizing against any housekeeping genes available. Positive controls and negative controls will be included in each analysis run.

Eligibility Criteria:
The inclusion criteria for the study group are:

1. All patients between the aged of 18 to 40 years.
2. Women diagnosed with polycystic ovarian syndrome fulfilling two out of three Rotterdam criteria (ie, oligo- or anovulation, hyperandrogenism, and polycystic ovaries on ultrasound).
3. Healthy volunteer with confirmed fertility (having at least one child previously), with a normal level of basic reproductive hormones and regular interval of the menstrual cycle (26 to 32 days). (Control group)

The exclusion criteria are:

1. Smoking
2. any hormonal treatment or other drugs to treat anovulation for at least 3 months before the study
3. Pregnancy or lactation during the preceding 12 months
4. Systemic disease such as endocrine or eating disorders besides PCOS
5. Diagnosed with a uterine or ovarian disease.
6. Any regular medication such as hormones, herbal substance, statins or corticoids for at least 3 months before the study.
7. History of intrauterine device placement.

The inclusion criteria for the control group are:

1. All patients between the aged of 18 to 40 years.
2. Healthy volunteer with confirmed fertility (having at least one child previously), with a normal level of basic reproductive hormones and regular interval of the menstrual cycle (26 to 32 days).

The exclusion criteria are:

1. Smoking
2. any hormonal treatment or other drugs to treat anovulation for at least 3 months before the study
3. Pregnancy or lactation during the preceding 12 months
4. Systemic disease such as endocrine or eating disorders besides PCOS
5. Diagnosed with a uterine or ovarian disease.
6. Any regular medication such as hormones, herbal substance, statins or corticoids for at least 3 months before the study.
7. History of intrauterine device placement.