The Effect of Omega-3 on the Number of Retrieved Ova , Fertilization Rate , and Embryo Grading in Subfertile Women Undergoing Intracytoplasmic Sperm Injection

This study aims to evaluate the role of preconceiving omega 3 polyunsaturated fatty acids supplementation in enhancing the proportion between follicles and retrieved ova, the fertilization rate, and the embryonic grading in subfertile females experiencing intracytoplasmic sperm injection management protocols. One-hundred twenty subfertile women aged 20-40 years-old undergoing intra-cytoplasmic sperm injection were recruited in this randomized double-blinded placebo-controlled clinical trial, at at Fertility Center/ Al-Sadr Teaching Hospital/ Al Najaf/ Iraq. They were randomly assigned into two groups; group A (omega-3) includes 60 subfertile women who received one capsule 1000mg omega-3 and Group B (placebo) includes 60 subfertile women who received a placebo contain Liquid Paraffin 500mg for eight weeks. The number of follicles, number of oocytes, fertilization rates, and embryonic quality were recorded in both groups. The study result revealed that the ratio of follicle/retrieved oocyte, the number of metaphase II oocytes, fertilization rate, and grade I embryo were more in the group A compared to group B. Supplementation with Omega-3 polyunsaturated fatty acids can increase the ratio of follicle/retrieved oocyte, the number of metaphase II oocytes, fertilization rate, and grade I embryo, and thereby improving the pregnancy outcome in intracytoplasmic sperm injection cycles.

Subfertility is a multifactorial disease that affects about 10-15 % of reproductiveaged couple's worldwide [1][2][3] .In Iraq the Primary Infertility was account for about 65.3% and Secondary Infertility 34.7% with ovulation disorders as the main cause of female infertility (41%) while (5%) for tubal obstruction, and only.Anovulatory problem is the 15% of the cases had unexplained infertility while abnormality in seminal fluid are the main causes in males [4][5] .Some of these fertility problems can be modified while others cannot be changed like age,several studies have reported that chances of getting pregnancy decrease rapidly every year after age 35years [6][7][8] .Various factors have a remarkable influence on the reproductive capability including changes in lifestyle, level of physical activity, nutrition, and overweight [10][11] .Carbohydrates, lipids, and proteins are involved in a wide range of physiological and biochemical activities of the human body.Fatty acids taking a particular interest, which has been linked with improved fertility in both spontaneous and assisted reproduction treatment conceptions 11 .
The polyunsaturated fatty acids play an important role as a cell membrane component, source of energy, and as signaling molecules 12 .
Omega-3 fatty acids (omega-3 FA) are precursors of hormones that are produced locally as eicosanoids that are important in the prevention and cure of multiple diseases, especially in women [17][18][19] .In general, eicosanoids derived from-6 PUFA are pro-inflammatory, while those coming from omega-3 are anti-inflammatory [20][21] .
Omega-3 can influence reproductive processes by acting as precursors for prostaglandin synthesisaswell; omega-3 can modulate the expression of many enzymes involved in prostaglandin and steroid metabolism which are found to be necessary for both ovarian and uterine function and seem to be essential in any successful reproductive procedure [22][23][24][25] .This study aims to investigate the possible effect of preconceiving supplementation of omega-3 PUFAs on intracytoplasmic sperm injection outcomes on the basis of the ratio between the follicles and retrieved oocytes, the rate of fertilization, and quality of the embryo.

Study Design
The current study is a prospective randomized double-blind Placebo-controlled clinical trial, performed at the Fertility Center in Al-Sadr Medical Teaching Hospital, Najaf Al-Ashraf, Iraq, fromJanuary 2017 to February 2018.The study was approved by the Iraqi Medical Ethics Committee in the University of Kufa, Najaf Al-Ashraf/ Iraq.

Study Population
One-hundred twenty subfertile women were randomly selected from subfertile women referred to Al-Sadr Teaching Hospital/ Fertility Center/ IVF Department in Najaf Al-Ashraf City.All women received adequate counseling and signed an informed consent after explanation of the research study aims and steps.Two women were excluded because they did not meet the inclusion criteria and three other did not complete the study.The recruited subjects were randomly assigned into two groups; the Omega 3 group (59 subfertile women) and Placebo group (56 subfertile women).

Exclusion Criteria
Women who have medical disorders e.g., diabetes mellitus, hypertension or thyroid diseases, sperm collection from epididymal aspiration, Percutaneous Epididymal Sperm Aspiration (PESA) and Testicular Sperm Extraction (TESE), frozen sperm, Consumption of any medications in the last 12 weeks that may influence hormonal assay, history of any diet in the previous three months, tobacco and alcohol consumption, and supplementation of n-3 PUFAs in the past three months.

Baseline Investigations
Blood pressures, random blood sugar, and thyroid-stimulating hormone all were measured before women's recruitment to the study for the exclusion of hypertension, diabetes mellitus,and thyroid dysfunction respectively.

Omega-3 and Placebo
The women of group A were given omega-3 for eight weeks, while the women of group B were given a placebo for eight weeks before starting the ICSI protocol.The dose of omega-3 was 1000mg one capsule every day composed of 180 mg eicosapentaenoic acid (EPA) and 120 mg docosahexaenoic acid (DHA), the placebo capsule contains 500mg paraffin manufactured by Alzahravi Pharmacology Company.The placebo and omega-3 capsules look similar and cannot be distinguished from each other (same form of package, same shape, same size and color of the capsule).All recruited subjects were followed-up every week by phone messaging, calls and were advised to return the drug pack every one-month visit.At one month, another pack with 30 capsules was supplied to the women.Neither the researcher nor women knew which group takes then-3 or placebo till the end of the research.After eight weeks, all women on day 2 of their cycle will be enrolled in controlled ovarian hyperstimulation depending on standard protocols that are used in fertility center.

Demographic Data Recording, Physical Examination,and Investigation
After recording the routine information, concerning the name, age, address, body weight and height for every woman included in the study, a careful history and physical examination were performed.Transvaginal sonography was performed on day 2 of the cycle to assess endometrial thickness, antral follicle count, and for the exclusion of any uterine or ovarian pathology.Serial transvaginal ultrasound was done for women every 2-3 days till signs of ovulation or ovum pickup for assessment of follicular size and maturation, and for observation of endometrial thickness measurement depending on the day of the cycle.

Hormonal Assays
Hormonal assays were performed, on day 2 ofthe cycle before starting stimulation protocol, 5ml of blood were taken for every woman participated in the study for Follicle Stimulating Hormone (FSH), Luteinizing Hormone (LH), Estradiol (E2), Prolactin and Thyroid-Stimulating Hormone (TSH) assay.Two days before Human Chorionic Gonadotropin (hCG) administration, other blood samples were collected to measure serum levels of E2 in the same way, 14 days after embryo transfer,a blood sample was collected to measure serum b-hCG level.All Hormonal measurements were done by Mini-vidus technique (using Kit from BioMerieux SA company/ France).

Ovarian Stimulation Protocols
Two ICSI stimulation protocols were used in this study; the stimulation protocol was selected according to individual patient characteristics.

Short Agonist Protocol (n=42)
On the cycle day 2, Gonadotropins started with Decapeptyle 0.1mg/day.After down-regulation of pituitary GnRH receptors has occurred, the dose of the GnRH agonist is reduced, and the ovulation induction is initiated with recombinant human Follicle Stimulating Hormone (Gonal-f, Merck Serono Specialities Pvt.Ltd, Italy) which contain Follitropinealfa 75 IU/ampoulethese are given subcutaneously, GnRH agonist continued till day of hCG injection.

GnRH Antagonist Protocol (n= 73)
The women received recombinant human Follicle Stimulating Hormone (Gonal-f, Merck) 75 IU/ampoule by daily subcutaneous (SC) injection.GnRH antagonist (Cetrotide 0.25 mg) was SC injected when the diameter of the leading follicle was 14-15 mm (Flexible regime) it administered continuously until the day of hCG administration.The dose of recombinant human Follicle Stimulating Hormone ranged between 150 and 450 IU, based on BMI, the age of the patient, and the anticipated ovarian response and it optimally adjusted depend on the transvaginal ultrasound reports for the number and size of growing follicles.

Ovum Pick Up (DOPU)
O v u l a t i o n i s i n d u c e d b y h C G administration (intramuscular Pregnyl 10000 I.U.Serono S.P.H, Italy) or by two ampoules (0.1mg) of Decapeptyle (Triptorelin) when the level of E2 >400pg/ ml and two or more follicles reached 17 mm diameter.Then oocytes retrieval was performed under vaginal ultrasound guidance using a transvaginal probe with an ultrasound-guided needle.
The seminal fluid sample was collected at the day of ovum pick up in a private room near the laboratory, after a minimum of 2 days and a maximum of 7 days of sexual abstinence into a clean, wide-mouthed container made of glass or plastic, The eggs are counted by an embryologist in the inverted microscope and stages of oocytes maturity are recorded.Before this done, denudation of the oocytes was performed.Germinal vesicle (GV) and metaphase I (MI) oocytes are excluded and only metaphase II oocytes (MII) are injected with Intracytoplasmic sperm injection.
The fertilization rate was defined as the ratio between the numbers of 2 pronuclei (PN) zygotes to the number of injected MII.FR = (NO. of 2PN on 1st day)/(Total NO.of injected oocytes)× 100.Each embryo was individually evaluated under the microscope early in the morning of day-2.Embryos were graded based on the following table : The most viable zygotes have (PN) of the same size and that is centrally located, with those which have nuclear percursor body of same size and number aligned at the pronuclear interphase

Primary and Secondary Endpoints
Primary endpoint is the embryo quality based on morphological classification as shown in Table (1).

RESULTS
Ta b l e ( 2 ) d i s p l a y s t h e g e n e r a l characteristics of subfertile women who have undergone ICSI.The mean age of studied subjects was 29.37 ± 5.51 years (20-39 years), the BMI means of the studied group was 27.84 ± 2.44 kg/ m2 (21.7-34.6 kg/m 2 ) and the mean duration of subfertility was 7 ± 3.21 years (2-19 years).
There were no statistically significant differences in the mean of age, BMI, , level of the studied hormones and duration of infertility between the omega-3 group and placebo group as shown in Table (3).
Comparison between Omega-3 and Placebo Group Regarding the Number of Follicles, Ratio of Follicles to Oocyte Retrieval, and the Ratio of Follicles No/ No of Injected Oocyte.
The numbers of follicles to the number of injected oocytes in the Omega-3 group were higher than that in the placebo group.However, the difference was not significant (p > 0.05) as shown in Figure (2).
The result of the study has shown that the fertilization rate and 2 Pronuclei (2PN) was higher in omega 3 group in comparison with the placebo group (p < 0.05) as shown in (Figure 3).Out of the (489) oocytes injected in the Omega-3 group, (303) were fertilized, whereas only (202) out of (422) injected oocytes in the placebo group were fertilized.Higher rates (p < 0.05) were observed in the Omega-3 group in comparison to the placebo group.
The total number of embryos in the omega-3 group was 6.05±0.47 and in placebo groups 5±0.41 as shown in Table (4).This improvement is the result of the increased number of MII oocytes in the Omega-3 group.However, Omega-3 has not only increases the number of  4), but it has also improved embryo quality and this is obvious a the number of grade 1 embryo 2.16±0.28 in the Omega-3 group while it is 0.67±0.13 in Placebo group as shown in Table (4).The total pregnancy rate (positive pregnancy test) in both groups was 35 women (30.4%) while 80 women had a negative pregnancy test.Out of 59 Women in Omega-3 group, 20 (33.9%) women developed pregnancy where in placebo group only 15(26.8%)women had positive pregnancy tests.However, the differences were statistically not significant as shown in Table (5).
Table (6) was done to exclude the effect of ovarian stimulation protocols, as 73 subfertile women undergoing antagonist protocol while only 42subfertilewomen in placebo but there was no significant difference on their effects on both studies groups.

DISCUSSION
The current study was conducted to clarify the impact of omega 3 PUFAs supplementation on ICSI clinical outcomes, by comparing the main ICSI parameter like the number of retrieved ova,  the fertilization rate, and the embryonic grading in two groups of subfertile women experiencing ICSI management protocols.One group receives omega 3 PUFAs supplementation and the other group receiving placebo for 56 days (8 weeks).The study attempts to ameliorate the effects of certain important confounders that may affect the result like BMI and age of participant by selecting the subfertile women with younger age and canceling obese women from the study and by matching such variables between the two groups 26 .
Fifty-seven (96.6%) out of 59 subfertile women in the Omega-3 group have their 1 st ICSI trial, while 55 (98.2%) out of 56 subfertile women in the placebo group were with 1st ICSI trial.Only 3 women have the second trial of ICSI.There were no significant differences between the two groups regarding the number of ICSI trial.This finding probably excludes the effect of a number of trials on ICSI results.Sixteen (27.1%) out of 59 subfertile women in the Omega-3 group follow agonist protocol and 43(72.9%)follow antagonist protocol, wherein Placebo group 26(46.4%)women follow agonist protocol and 30(53.6%)women follow antagonist protocol.The results of our research study were in harmony with other research studies that concluded the antagonist protocol was shown to be an easy and safe protocol [27][28][29] .
Regarding the type and causes of infertility high percentage of subfertile cases presented with primary subfertility 107(93%) and only 8 (7%) with secondary subfertility, male factor carried a higher percent 102 (88.7%) than other causes that indicated for ICSI.This finding supported by other research studies who found that male factor of subfertility is predominant than other causes (56.43%) [30][31] .The possible explanation for the high incidence of infertility among Iraqi men is that our country environment suffered from many profanations as result of many wars, and males tend to be exposed more directly to hazards of different type of Chemical Warfare Agents as a participates in the wars.
Despite the pregnancy rate reflects the success of the ICSI process, it is better to use live birth rate better to use live birth rate but this needs more time and follow up.For this reason, we considered the embryo quality to be the primary endpoint.
The numbers of follicles in the omega-3 group were significantly higher than that in the Placebo group, and there is a higher number of the retrieved oocyte in the Omega-3 group these findings agree with other findings in prior studieswho reported a high intake of dietary fish oil led to a significant increase in the ovulation and follicles development 33 .
The result of this study also revealed a significant increase in the numbers of the embryo in the Omega-3 group in comparison with the Placebo group.A possible explanation may be due to the fact that PUFAs supplementation associated with suppression of production of PGF2 by compacting with prostaglandin endoperoxide synthase (PGHS) enzyme that required for the change of the Arachidonic acid to the PGF2.It is believed that decreased PGF2 level, elevating the chance of preserving the life of the newly developed embryo [34][35][36] .This agrees with Hammicheand Kermack they reported that assessing preconception supplementary n-3 PUFA that results in improving embryo morphology and ART outcomes [37][38] .

CONCLUSION
T h e s e f i n d i n g s s u g g e s t t h a t supplementation with Omega-3 FAs in IVF techniques could positively influence the final result of the reproductive sequel.However further research studies are required considering racial and ethnic differences response to omega-3 FAs in order to permit future Meta-analysis performance and application in clinical guidelines of management.

Fig. 2 .Fig. 3 .
Fig. 2. Comparisons between Placebo and Omega 3 Group Regarding the Number of Follicle and Number of Injected Oocyte

Table 2 .
Mean and Standard Error of the General Characteristics of the all Participants in the Study (n=115)

Table 3 .
Comparison between Omega 3 and Placebo Groups in Terms of Age, BMI, and Duration of Infertility

Table 4 .
Comparison between Omega3 and Placebo Group Regarding's ICSI Outcome Chicago, IL, USA).Values were expressed as mean ±SE.A P-value < 0.05 was considered to be statistically significant, P. value <0.01 was considered to be statistically highly significant.

Table 5 .
Association between Groups and Pregnancy Test Result

Table 6 .
Association between Pregnancy rates and Controlled Ovarian Hyperstimulation Protocol