337J Contemp Med Sci | Vol. 8, No. 5, September-October 2022: 337–342 Original Relation of Serum and Follicular Level of BMP15 with Oocyte Quality, Embryo Grading and Pregnancy Rate Zainab Hassan Hashim1*, Lubna Amer2, Estabraq A. Al-Wasiti3 1Department of Clinical Reproductive Physiology, High Institution for Infertility Diagnosis and Assisted Reproductive Technologies, University of Al-Nahrain, Baghdad, Iraq. 2Institution for Infertility Diagnosis and Assisted Reproductive Technologies, University of Al-Nahrain, Baghdad, Iraq. 3Department of Biochemistry, College of Medicine, University of Al-Nahrain, Baghdad, Iraq. *Correspondence to: Zainab Hassan Hashim (E-mail: zainab.hassan@ierit.nahrainuniv.edu.iq) (Submitted: 03 March 2022 – Revised version received: 21 March 2022 – Accepted: 07 April 2022 – Published Online: 26 October 2022) Abstract Objectives: Use of serum and follicular fluid concentration of oocyte secreted factors BMP15 as biomarkers of oocyte quality, embryo quality and it’s relation to pregnancy rate. Methods: Eighty eight women were included in this study; they were selected from those undergoing intra-cytoplasmic sperm injection. Results: Positive pregnancy was achieved by 14 women accounting for 19.0% (total number of women that reach embryo transfer was 72). No significant difference in mean serum BMP15 between pregnant and non-pregnant women, but the level of follicular fluid BMP15 was significantly higher in pregnant women. MI oocyte count was not significantly correlated to serum or follicular fluid BMP15 (P > 0.05). MII oocyte count showed highly significant positive correlation to serum and follicular fluid BMP15 (P < 0.01). Grade 1 embryo count showed highly significant positive correlation to serum and follicular fluid BMP15 (P < 0.01), Grade 2 embryo count showed significant positive correlation to serum BMP15 (P = 0.032), but the grade 2 embryo count showed non-significant correlation to follicular BMP15 (P > 0.05). Also grade 3 embryo count showed non-significant correlation to serum and follicular BMP15 (P > 0.05). Conclusion: The current study revealed that serum and follicular BMP15 could be used as indicator for oocyte maturity, and serum BMP15 could be used as indicator of good quality embryos. Keywords: Follicular level, BMP15, oocyte, embryo grading, pregnancy rate ISSN 2413-0516 Introduction Infertility is defined as the inability, of a couple to have preg- nancy after a period of one year, in those women under 35 years of age or after 6 months in those women above 35 years of age, in spite of regular (3 to 4 times/week), adequate and unprotected sexual intercourse. The key limiting factor in female fertility is the oocyte quality, and till now there is poor understanding of what factors that determine the oocyte quality or the mechanisms that governing it.1,2 About 35% of infertility cases are caused by female fac- tors, 35% related to male factors, 20% caused by both male and female factors, and 10% by unknown causes.3 The key limiting factor in female fertility is the oocyte quality; the quality of oocyte greatly affects early embryonic survival, also establishment with maintenance of pregnancy, devel- opment of the fetus, and even causes some adult diseases.1 Bone morphogenetic protein 15 (BMP15) and growth dif- ferentiation factor 9 (GDF9), have a unique feature, within the transforming growth factors-b super-family is that the expression of the protein is essentially restricted to the gam- etes (oocyte). BMP15 and GDF9 are expressed in the oocyte during folliculogenesis, from the earliest stages.4 They are expressed in high levels by the oocyte throughout follicu- logenesis, so they are could be regarded a good indicator for oocyte quality, and measuring them in the serum which is rapid, non-invasive and easy test could give a great clue to female fertility.5 This study aimed to use serum and follicular fluid concen- tration of oocyte secreted factors BMP15 as biomarkers of oocyte quality, embryo quality and then to study it’s relation with the pregnancy rate. Materials and Methods A prospective study was conducted in the High Institute of Infertility Diagnosis and Assisted Reproductive Technolo- gies, Al-Nahrain University, from Nov., 2020 to July 2021. One hundred and seventy six women were included in this study. The study subjects involved eighty eight women who were selected from those attended the High Institute for Infertility Diagnosis and Assisted Reproductive Technologies, they were enrolled in IVF program. Inclusion Criteria •    All couples undergoing IVF/ICSI protocols. •    Women at any age from 18 to 47 years old. •     Infertility due to female factors: tubal blockage, unovula- tory cycles, and mild-moderate cases of endometriosis that diagnosed laproscopically. •    Couples with male factor infertility. •    Unexplained infertility. Exclusion Criteria •     All  types  of  congenital  anomalies  of  the  reproductive  system. •    Uncontrolled systemic and endocrine disorders. •    Women with BMI more than 30 kg/m2 Methods and Study Design A total of eighty eight patients undergoing IVF/ICSI cycle were  evaluated: •     Taking  full  obstetrical,  medical,  surgical  history  with  assessment of weight and height to obtain (BMI). mailto:zainab.hassan@ierit.nahrainuniv.edu.iq 338 J Contemp Med Sci | Vol. 8, No. 5, September-October 2022: 337–342 Relation of Serum and Follicular Level of BMP15 with Oocyte Quality, Embryo Grading and Pregnancy Rate Original Z.H. Hashim et al. •     Examinations of the woman clinically and gynecologi- cally to check for any abnormality. •     For  male  partners,  the  seminal  fluid  analysis  was  assessed according to WHO 2010.  •     Doing analysis of female hormones (LH, FSH, E2, Prol- actin, Testosterone and TSH) at the second day of the  menstrual cycle. •     All women were enrolled to only one type of controlled  ovarian  hyperstimulation  (COH)  protocols  which  is  Gonadotropin releasing hormone antagonist protocol. •     Follow up of the patients by doing serial vaginal ultra- sound and doing serum level of estradiol (E2) and then  accordingly to the result, ovum pick up done. •     Oocyte  retrieval  done  with  guidance  of  trans-vaginal  ultrasound  after  ovulation  trigger  with  HCG  about  (35–36) hrs. •     At the day of ova pick up, serum and follicular fluid sam- ples were obtained from each woman for measurement of BMP15. The antagonist protocol involved ovarian stimulation with gonadotropins since the second day of the menstrual cycle fol- lowed by the administration of a GnRH antagonist (Cetrorelix  acetate for injection 0.25 mg: Cetrotide®, Merk, Switzerland),  using flexible method and given when the size of the largest  follicles reach (13–14) mm. The initial dose of FSH was 75–  300 IU daily according to patient condition. With serial vag- inal U/S for checking the number and size of ovarian follicles  and  for  the  endometrial  thickness  (ET),  in  addition  serum  level of Estradiol (E2) was done. The serum level of (E2) Estra- diol was measured at day of ovulation triggering by (HCG)  administration. The oocyte grading was at retrieval could be either imma- ture oocyte and this is called Germinal vesicle (GV), in which the corona and cumulus cells, are tightly packed around the oocyte, with presence of circular structure inside it, that is called the (germinal vesicle), the other immature oocyte is called Metaphase I (MI). The mature oocyte is called Meta- phase II (MII) which has polar body. MI oocyte characterized  by the absence of a polar body or a germinal vesicle, and it is intermediate stage between the GV and MII (mature) stages. ICSI Processes The aspirated follicles were examined at the IVF laboratory, in petri dish immediately. Flushing was done then kept 1–2 hrs in the (37°C/ CO2) incubator, all oocytes after that were subjected to denudation and grading in a Laminar Flow Cabinet. The  mature eggs were selected by a specialized pipette, and by a  very delicate, sharp and hollow needle which is used to held, immobilize and then pick up a single sperm. After that, the  sperm was inserted by the needle carefully through egg shell into its cytoplasm. Then the eggs were kept in the CO2 incu- bator and carefully monitor the result of cell division, by using Nikon ICSI Microscope. Embryo Quality and Grading Zygotes after insemination, were observed after (18–20) hours  to check for the presence of (2) pronuclei and after (25–29) hours to observe the presence of early cleavage, which is con- sidered a sign of better implantation rates. The presence of 2 pronuclei at day 1, was regarded as a good prognostic sign. Then at day two (43–45 hours after insemination) and day three  (67–69  hours  after  insemination)  the  embryos  were  evaluated. Good quality embryos were considered when they were homogeneous, with normal kinetics (4) cells at day 2 and (7–9)  cells  at  day  3,  and  containing  <10%  of  cytoplasmic  fragments. The embryos at the third day, were classified as being with or without compaction, which referred to all embryos that underwent the compaction process, the embryos could be at the beginning of compaction when the fusion of the mem- brane was visible, in this stage the counting of the number of cells is still possible, and those embryos with full compaction, in those embryos the distinguishing of cell boundaries was not possible. Embryo Transfer: The dividing embryos were then replaced into the uterine cavity under pelvic ultrasound guid- ance and by an embryo transfer catheter. Results The pregnancy rate in infertile women enrolled in the current study is shown in Figure 1. Positive pregnancy was achieved by 14 women accounting for 19.0%. Total number of patient was 88,  a  number  of  cases were not  included  in  counting  preg- nancy rate this included five cases of empty follicles, four cases of embryonic developmental arrest, six cases of failed fertiliza- tion and one patient refuse embryo transfer. So the number of  cases that were included in counting pregnancy rate was 72  patients. The characteristics of infertile women enrolled in this study are shown in Table 1. The mean age of all enrolled women 32.25 ± 6.41 years and the mean age of women with positive pregnancy was significantly lower than that of non-pregnant  women  (29.14  ±  4.54)  years  versus  (32.76  ±  6.55) years, respectively (P = 0.050). The mean duration of infertility of all enrolled women was (7.89 ± 3.87) years and  the mean duration of infertility of pregnant women was lower than that of non-pregnant women (6.93 ± 3.08) years versus  (8.05 ± 3.98) years; however, the difference did not reach sta- tistical significance (P = 0.319). Out of all enrolled women, primary infertility was seen in 65 (74.0%) women, whereas,  secondary infertility was seen in 23 (26.0%) women and there was no significant difference in the frequency distribution of women according to type of infertility with respect to preg- nancy outcome (P = 1.000). The mean BMI for pregnant women  was  (26.71  ±  2.60),  and  for  non-pregnant  women  (26.72 ± 3.01), there was no significant difference in the fre- quency distribution of women according to BMI with respect to pregnancy (P = 0.958). Fig. 1 Pie chart showing pregnancy rate of women undergoing ICSI. 339J Contemp Med Sci | Vol. 8, No. 5, September-October 2022: 337–342 Z.H. Hashim et al. Original Relation of Serum and Follicular Level of BMP15 with Oocyte Quality, Embryo Grading and Pregnancy Rate Table 2. BMP15 serum and follicular levels and it’s relation to pregnancy rate Characteristic Total N = 72 Positive pregnancy N = 14 Negative pregnancy N = 58 P Serum BMP15 Mean ± SD 157.89 ± 51.08 169.79 ± 23.22 155.95 ± 54.13 0.350 I NSRange 14–296 130–213 14–296 Follicular fluid BMP15 Mean ± SD 149.64 ± 38.86 179.71 ± 29.44 144.74 ± 38.12 0.001 I HSRange 24–253 121–253 24–197 N, number of cases; SD, standard deviation; I, independent samples t-test; NS, not significant at P > 0.05; HS, highly significant at P ≤ 0.01. Table 1. Characteristics of infertile women enrolled in this study Characteristic Total N = 72 Positive pregnancy N = 14 Negative pregnancy N = 58 P Age (years) Mean ± SD 32.25 ± 6.41 29.14 ± 4.54 32.76 ± 6.55 0.050 I SRange 20–47 23–40 20–47 Duration of Infertility (years) Mean ± SD 7.89 ± 3.87 6.93 ± 3.08 8.05 ± 3.98 0.319 I NSRange 1–17 2–12 1–17 Type of infertility Primary, N (%) 65 (74.0%) 10 (71.4%) 55 (74%) 1.00 Y NSSecondary, N (%) 23 (26.0%) 4 (28.6%) 19 (26%) BMI (kg/m2) Mean ± SD 26.72 ± 2.81 26.71 ± 2.60 26.72 ± 3.01 0.958 I NSRange 20.44–30.75 21.46–30.75 20.44–30.70 N, number of cases; SD, standard deviation; I, independent samples t-test; Y, Yates correction for continuity; NS, not significant at P > 0.05; S, significant at P ≤ 0.05. Relation of Pregnancy Rate to Serum and Follicular Level of BMP15 At day of ova pick up the bone morphogenetic protein 15 (BMP15) serum and follicular levels are shown in Table 2. There was no significant difference in mean serum BMP15 (169.79  ±  23.22)  versus  (155.95  ±  54.13)  between  pregnant  and non-pregnant women. But the level of follicular fluid BMP15 was higher in pregnant women in compression with non-pregnant women in a highly significant manner (179.71 ±  29.44) versus (144.74 ± 38.12) (P = 0.001). Receiver Operating Characteristic (ROC) Curve Analysis to find the Cutoff Value of BMP15 that can Predict a Positive Pregnancy Outcome Receiver  operating  characteristic  (ROC)  curve  analysis  was  carried out to find the cutoff value of BMP15 that can predict a positive pregnancy outcome and the results are shown in Figure 2 and Table 3. The cutoff value of BMP15 was > 129 but with poor accuracy (56.6%) since the area under curve (AUC)  was less than 0.7.  The Correlations of Serum and Follicular Fluid BMP15 to Oocytes Maturity The correlations of serum and follicular fluid bone morphoge- netic protein 15 (BMP15) that measured at day of ova pick up to oocytes maturity are shown in Table 4. MI oocyte count was not significantly correlated to serum (0.195) or follicular fluid BMP15 (–0.005). MII oocyte count showed highly significant positive  correlation  to  serum  (0.270)  and  follicular  fluid  BMP15 (0.413) (P < 0.01).  The Correlations of Serum and Follicular Fluid BMP15 to Embryo Grading The correlations of serum and follicular fluid bone morphoge- netic protein 15 (BMP15) to embryo grading are shown in Table 5. Grade 1 embryo count showed highly significant pos- itive correlation to serum (0.273) and follicular fluid (0.301)  BMP15 (P < 0.01), Grade 2 embryo count showed significant  positive correlation to serum BMP15 (0.215) (P = 0.032). While grade 2 embryo count showed non-significant correla- tion to follicular BMP15 (0.133) (P > 0.05). Also grade 3 embryo count showed non-significant correlation to serum (–0.099) and follicular (–0.043) BMP15 (P > 0.05). The Correlations of Serum BMP15 to Peri- follicular and Endometrial Blood Flow Doppler The Resistive Index (RI) and pulsatility index (PI) of perifolli- cular blood flow measured by pulsed Doppler ultrasound at the day of ova pickup showed no significant correlation to serum BMP15, RI (0.312), PI (0.309) (P > 0.05). 340 J Contemp Med Sci | Vol. 8, No. 5, September-October 2022: 337–342 Relation of Serum and Follicular Level of BMP15 with Oocyte Quality, Embryo Grading and Pregnancy Rate Original Z.H. Hashim et al. Table 3. The results of receiver operating characteristic (ROC) curve analysis to find the cutoff value of BMP15 that can predict a positive pregnancy outcome Characteristic BMP15 Cutoff >129 AUC 0.566 95% CI 0.463 to 0.664 P-value 0.350 NS Sensitivity % 100.0 Specificity % 22.1 Accuracy % 56.6 AUC, area under curve; CI, confidence interval; NS, not significant. Table 4. Correlations of serum and follicular fluid BMP15 oocytes maturity Characteristic Correlation Index Serum BMP15 Follicular fluid BMP15 Immature metaphase I (MI) oocytes R 0.195 –0.005 P 0.052 0.962 Mature metaphase II (MII) oocytes R 0.270 0.413 P 0.007* <0.001* *, highly significant at P ≤ 0.01. Table 5. The correlations of serum and follicular fluid BMP15 to embryo grading Characteristic Correlation Index Serum BMP15 Follicular fluid BMP15 Grade 1 embryo R 0.273 0.301 P 0.006** 0.002** Grade 2 embryo R 0.215 0.133 P 0.032* 0.188 Grade 3 embryo R –0.099 –0.043 P 0.089 0.114 *, Significant at P ≤ 0.05; **, highly significant at P ≤ 0.01. Fig. 2 Receiver operating characteristic (ROC) curve analysis to find the cutoff value of BMP15 that can predict a positive preg- nancy outcome. Also the Resistive Index (RI) pulsatility index (PI) of perifollicular blood flow measured by pulsed Doppler ultrasound at the day of ova pickup showed non-significant correlation  to  follicular  BMP15,  RI  (–0.068),  PI  (–0.110)  (P > 0.05). Furthermore the Resistive Index (RI), pulsatility index (PI) of endometrial blood flow measured by pulsed Doppler ultrasound at the day of embryo transfer showed no significant correlation to serum BMP15, RI (–0.002), PI (0.202) (P > 0.05). Also showed non-significant correlation to follicular BMP15, RI (–0.143), PI (0.034) (P > 0.05), as shown in Table 6. Discussion The main aim of this study was to find an easy, fast, not expen- sive and available outpatient test to be an indicator for female fertility. Various studies regarding AMH, FSH, LH, prolactin, E2  and other hormones beside genetic variants investigations have been performed and obtained as a good and specific bio- markers determined in last decade. All of these efforts were used to predict female reproductive potential and they used for different female sexual activities and are investigated with their receptors in different female sexual system disorders affected.6-10 These hormones are used to estimate growing fol- licles number in the ovary and to estimate and predict the response of ovaries to stimulation by gonadotropin. These bio- markers provide only an indirect evaluation of oocyte func- tion and yield no information about quality of the oocyte, because they are not derived from the oocyte itself.11 BMP15 is known to be secreted only by the oocyte, essen- tial for process of folliculogenesis, quality of the oocyte and female fertility, so these factors could be regarded as oocyte function biomarkers.12 Positive pregnancy was achieved by 14 women accounting for 19.0%. The rate was low when compared with other studies like a study done by De Geyter et al. that found that pregnancy rate was 28%,13 also other study done by Jassim WH, et al.  found pregnancy rate to be 25.4%.14 The pregnancy rate was low because 4 cases of testicular biopsy, 2 cases of moderate endometriosis and also there were 14 case with age above 40 years were included in the this study, furthermore the SARS-CoV-2 (Covid-19) pandemic also might be one of the  causes of decrease in pregnancy rate, this is supported by study result done by Maya, W. D. C, et al., that found that germ cell  destruction and testicular damage was clearly observed in patient  with  Covid-19,15 and the testes that infected with SARS-CoV-2-showed extensive peritubular fibrosis, vascular  congestion with extensive destruction of germ cell.16 Further- more,  SARS-CoV-2  could  cause  ovarian  tissue  damage  and  decline in the function of the ovary and oocyte quality, causing female infertility and may cause miscarriage.16 A number of cases were not included in counting pregnancy rate, this included five cases of empty follicles, four cases of embryonic developmental arrest, six cases of failed fertilization and one  patient refuse embryo transfer. So the number of cases that  were included in counting pregnancy rate was 72. 341J Contemp Med Sci | Vol. 8, No. 5, September-October 2022: 337–342 Z.H. Hashim et al. Original Relation of Serum and Follicular Level of BMP15 with Oocyte Quality, Embryo Grading and Pregnancy Rate Relation of Pregnancy Rate to Serum and Follicular Fluid Levels of BMP15 There was no significant difference in mean serum BMP15 between pregnant and non-pregnant women, but the level of follicular fluid BMP15 was higher in pregnant women in com- pression with non-pregnant women in a highly significant manner. But a study done by Li et al., on gene expression found  that BMP15 mRNA expression levels were closely associated with pregnancy outcomes.17 Many factors might affect preg- nancy rate other than oocyte quality like male factors for example abnormality in DNA as in sperm retrieved by testic- ular biopsy, also due to bad endometrial receptivity and psy- chological problems. Correlations of Serum and Follicular Fluid BMP15 to Oocyte Maturity The current study showed that MI oocyte count were not significantly correlated to serum or follicular fluid BMP15 (P > 0.05) Table 3. While MII oocyte count showed highly  significant positive correlation to serum and follicular fluid BMP15 (P < 0.01).  The result correspond to study result done by Li et al. on  gene expression found that the mRNA expression levels of BMP15 were closely related to maturation of the oocyte, fer- tilization  and  outcomes  of  the  pregnancy.17 Furthermore a study done by others stated that a beneficial synergistic effects are exerted by OSFs on the maturation of nucleus and cyto- plasm, rapid energy utilization and oxidative stress manage- ment.18,19 BMP15 is secreted by the oocyte in a primary follicle,  which,  together  organize  the  granulosa  and  theca  cells that surround the oocyte into oocyte–cumulus–follicle complex. The granulosa at this time secretes AMH, that affects the oocyte. Throughout the development of the fol- licle, this autocrine–paracrine dialogue between the somatic cells and the oocyte continues and is regarded essential for establishing the fertilization potential and oocytes develop- mental competency.20 The correlations of serum and follicular fluid and bone morphogenetic protein 15 (BMP15) to embryo grading are shown in Table 4. Grade 1 embryo count showed highly signif- icant positive correlation to serum and follicular fluid BMP15 (P < 0.01). Grade 2 embryo count showed significant positive  correlation to serum BMP15 (P = 0.032). This results were supported by study results of Canosa, S.,  et al., that found that the blastocyst group (BL) of embryos  showed faster kinetic in a significant manner, and the expres- sion of BMP15 mRNA was higher in CCs of this group with  significant value, as compared to arrested embryos.21 Also sup- ported by study done by Daneshjou, D. et al., that found that there is positive correlation between the expression level of BMP15  mRNA  with  the  fertilization  rate  and  grade  I  embryos.22 Conclusion Accordingly the observed data conclude that: 1.     Serum and follicular BMP15 could be used as indicator  for oocyte maturity. 2.     Serum  BMP15  could  be  used  as  indicator  of  grade  I  embryos. Conflict of Interest None.  Table 6. The correlations of serum and follicular BMP15 to perifollicular and endometrial blood flow doppler Characteristic Correlation index Perifollicular RI Perifollicular PI Endometrial RI Endometrial PI Serum BMP15 R 0.312 0.309 –0.002 0.202 P 0.207 0.211 0.989 0.217 Follicular fluid BMP15 R –0.068 –0.110 –0.143 0.034 P 0.788 0.782 0.397 0.839 References 1. Adhikari, D., Lee, I. W., Yuen, W. S., and Carroll, J. (2022). Oocyte mitochondria—key regulators of oocyte function and potential therapeutic targets for improving fertility. Biology of Reproduction, 106(2): 366–377. 2. Mustafa, M., Sharifa, A. M., Hadi, J., IIIzam, E., and Aliya, S. (2019). Male and female infertility: causes, and management. IOSR Journal of Dental and Medical Sciences, 18: 27–32. 3. Aghajanova, L., Hoffman, J., Mok-Lin, E., and Herndon, C. N. (2017). Obstetrics and gynecology residency and fertility needs: national survey results. Reproductive sciences, 24(3): 428–434. 4. Sanfins, A., Rodrigues, P. and Albertini, D. 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