Infertility

Ethiopian Journal of Reproductive Health (EJRH) July, 2021 Volume 14, No. 2 IN VITRO FERTILIZATION (IVF) OUTCOMES AND PREDICTING FACTORS IN A RESOURCE-LIMITED SETTING Thomas Mekuria, MD1, Lemi Belay Tolu, MD1, Mekitie Wondafrash, PhD2 ABSTRACT INTRODUCTION: Ethiopia has only one public In-vitro fertilization (IVF) center which was opened in 2019. The aim of the study is therefore to determine predictors of the outcome of IVF in the only public fertility center in Addis Ababa, Ethiopia. METHOD: The study is conducted in the public IVF center in Ethiopia between; April 01, 2019, to March 30, 2020. A retrospective cross-sectional study design was employed. All IVF clients meeting the inclusion criteria were included in the analysis. RESULT: There were a total of 199 couples included in the study. The clinical pregnancy rate was found to be 30.1%. The odds of getting pregnant is 61% less among participants with female partners age ≥35 years, AOR 0.39, CI 0.18-0.83 with a p-value of 0.015. Good responders ((≥4 oocytes retrieved) accounts for 152(76.4%) of the cases. Age of female partner, day 3 Follicle Stimulating Hormone (FSH), and Antral Follicle Count (AFC) count ≥5 were significantly associated with good ovarian stimulation response with a p-value of 0.050,0.002 and 0.005 respectively. CONCLUSION: Even though near two-thirds of the study participants did not know their exact date of birth, the reported age of female partner <35 years is associated with both good ovarian response and occurrence of pregnancy, emphasizing its importance for clinical decision making. Day 3 FSH and AFC ≥5 were associated with good ovarian stimulation response. Therefore, we recommend the combination of female partner age, day 3 FSH, and AFC ≥5 to predict ovarian response in low resource settings, since variables can be readily available without much cost to patients. Furthermore, we recommend follow up studies with a large sample size and prospective cohort design to appropriately compare the different predictors of ovarian response in our setting to develop evidence-based set up specific IVF protocols and guidelines KEYWORDS: Pregnancy rate, IVF, Resource limited setting, Predictors, Infertility, Ethiopia (The Ethiopian Journal of Reproductive Health; 2021; 14;1-10) 1 St. Paul’s Hospital Millennium Medical College, Addis Ababa, Ethiopia 2 St. Paul Institute for Reproductive Health &Rights, Addis Ababa, Ethiopia 1  Ethiopian Journal of Reproductive Health (EJRH) July, 2021 Volume 14, No. 2 INTRODUCTION et al. reported that AFC was the best and the only independent parameter to predict poor response, One in every four couples in developing countries but the study had limited value in predicting is affected by infertility1. Infertility, as such, is one pregnancy success20. In Ethiopia, there is no of the major public health problems identified published research on IVF as the service was not in Ethiopia. It is estimated that infertility affects available for the general public until recently. 15 to 20% of couples in Ethiopia2. Realizing the The aim of the study is, therefore, to determine magnitude of infertility in the country, Saint Paul’s outcomes and predictors of IVF among infertile Hospital Millennium Medical College (SPHMMC), couples managed at the SPHMMC CFRM, Addis in collaboration with the Ministry of Health, has Ababa, Ethiopia. opened the first public Center for Fertility and Reproductive Medicine (CFRM) with functioning METHOD AND MATERIALS IVF in the country, which was inaugurated in Study setting, period, and design February 2019. Before the inauguration of the A retrospective cross-sectional study design was center, only one private facility has been providing employed to recruit study subjects. The study was IVF services for over a hundred million population. conducted at the center for reproductive medicine In the last three decades, huge strides have been (CFRM) which is a fertility center administered made in terms of improving pregnancy and live under the SPHMMC department of obstetrics and birth rates for IVF cycles. Previous studies have gynecology. SPHMMC is a tertiary teaching referral established predictors of ovarian response such as hospital under the Federal Ministry of Health female age, antral follicle count (AFC), serum anti- (FMOH). The center is located in Addis Ababa, Mullerian hormone (AMH), serum FSH, and serum the capital city, and is the first public IVF center in LH concentrations3. Female fecundity signifcantly Ethiopia. Since its inauguration in February 2019, declines approximately at age 35 years and more more than 5000 infertile couples were evaluated, sharply after age 37 years4, and the chances of and more than 200 IVF cycles were provided in the successful pregnancy become very low after the center. All patients who underwent IVF at the center age of 405-7. Accurate prediction of ovarian for reproductive medicine (CFRM, SPHMMC) from response and establishing a tailored treatment April 01, 2019, to March 31, 2020, were included in strategy for those patients would improve the IVF this study, except couples who have frozen embryo outcomes 9-11. transfers. IVF outcomes were dependent variables. Basal follicle-stimulating hormone (FSH), anti- IVF outcomes were measures in terms of clinical Müllerian hormone (AMH), inhibin B, and antral pregnancy rate diagnosed by a serum pregnancy test follicle count (AFC), have been used to predict and ovarian response measured by the number of ovarian response and IVF outcomes. Basal FSH oocytes retrieved. Patients with ≥4 oocytes retrieved and inhibin B were found to have low predictive were called good responders to controlled ovarian power 12,13. On the other hand, AMH and AFC stimulation, while those with <4 oocytes were called seem to be the most reliable determinants of poor responders. The independent variables were ovarian response. Some investigators concluded age, AFC, BMI, AMH, and day 3 FSH. that the predictive accuracy of these two parameters Treatment protocol was similar14-17, whereas other studies have shown Treatment protocols included in the study were that AMH was superior to AFC in predicting conventional long protocol, antagonist protocol ovarian response18,19. However, there is a limited (short protocol), and minimal stimulation number of studies that assessed AFC as a predictor protocols. For long protocol, either highly purified of IVF outcomes in poor responders20-22. Mutlu urinary FSH (Fostimon 75 IU) or recombinant 2 Ethiopian Journal of Reproductive Health (EJRH) July, 2021 Volume 14, No. 2 FSH (Gonal F 75 IU), were used for stimulation non-parametric test of difference of means (Mann starting from day 3 of the cycle. The individual Whitney U test). Statistical significance was declared dose was calculated based on the age, BMI, and at p=0.05 and all tests were two-sided. A full model AFC of the client. Downregulation was achieved assessing the relationship between the pregnancy by depot Goserelin (Zoladex 3.6 mg) on the 21st test result and predictor variables was constructed day of the previous cycle. The menstrual cycles of after which non-significant variables were removed all women using this protocol were synchronized by by a backward procedure using the likelihood ratio the use of COCs (combined oral contraceptives). test (p<0.05) “ A variable antagonist protocol was employed where Ethical Considerations downregulation was started with Cetrotide 0.25 Ethical clearance and permission letter to conduct mg whenever the leading follicle/s reached 12cm the study and publish the outcome was obtained in diameter. For minimal stimulation protocol, from the Institutional Review Board (IRB) of Letrozole 5mg PO was started on day 2 of the SPHMMC. Confidentiality was maintained during cycle and continued for 5 days. On the 4th day, data collection, analysis, and interpretation by transvaginal ultrasound monitoring was done to avoiding recording of names and returning client assess the initial response and start hMG SC 150 records to their place after completion of data mg. Once the leading follicle attained 14 mm in collection. All the datasets used and/or analyzed size, down-regulation with Cetrotide was instituted. during the current study are included in the Trigger for all three protocols was decided if 3 or manuscript and available from the corresponding more leading follicles reached 18mm or more in author on reasonable request size or greater or equal to 5 follicles reach/exceed 16mm in size. RESULT Data collection and measurement There were a total of 208 couples who underwent Data were collected by two trained data collectors IVF during the study period. Out of these, 9 using pretested well-structured questionnaires. The women had their embryos frozen and did not medical record of patients who underwent IVF have fresh embryo transfer making the total fresh during the study period was identified from the embryo transfers during the study period 199. CFRM IVF registration. The records of fertility Semen analysis results were abnormal in 25% of care seekers were then reviewed, and the data male partners. Close to two-thirds of the women were collected using an open data kit (ODK). The in the study did not know their exact date of birth primary investigator supervised the data collection (64.5%). The median reported age was 32.5 years process on daily basis. (Table 1). Data processing and statistical analysis The data were on an open data kit (ODK) and were checked for completeness and then imported into Stata statistical software release 15 (StataCorp, College Station, TX, USA) for analysis. Univariate analyses were performed using proportions and means (standard deviation), or medians (interquartile range) when the distribution was not normal. The association of the pregnancy test result and independent variables were assessed using Fisher’s exact test, independent t-test, or the 3  Ethiopian Journal of Reproductive Health (EJRH) July, 2021 Volume 14, No. 2 Table 1: Baseline characteristics of the study subjects Factors associated with controlled ovarian stimulation _________________________________________ response Characteristics (n=208) No. % Good responders ((≥4 oocytes retrieved) accounts for __________________________________________ 152(76.4%) of the cases. Age of female partner, day 3 Date of birth known FSH, and AFC count ≥5 were significantly associated Yes 73.0 35.1 with good ovarian stimulation response (Table 2). No 135.0 64.9 Female partner body mass index Table 2: Bivariate analysis of factors associated with controlled <18.3 6.0 2.9 ovarian stimulation response 18.3-24.9 133.0 63.9 __________________________________________________________ Factor Good responders Poor responders P-value* 25-29.9 44.0 21.2 (≥4 oocytes retrieved) (<4 oocytes 30-34.9 21.0 10.1 n=152 retrieved) n=56 >34.9 4.0 1.9 _________________________________________________________ Marital status Age of female partner, 32.0 (29.0, 36.0) 35.0 (31.0, 37.0) 0.016 Married 208.0 100.0 median (IQR) Educational status of female partner Age of female partner No formal education 25.0 12.1 <35 years 95 (62.9%) 26 (47.3%) Completed primary level education 13.0 6.3 0.050 Completed secondary level education 70.0 33.8 ≥35 years 56 (37.1%) 29 (52.7%) Diploma and above 99.0 47.8 Female partner AFC 12.0 (8.0, 14.0) 7.0 (5.0, 10.0) <0.001 status, median (IQR) Male date of birth known Female partner AMH 0.9 (0.5, 2.6) 0.6 (0.4, 1.3) 0.110 Yes 30.0 14.4 median (IQR) No 178.0 85.6 Female partner 5.5 (3.9, 6.7) 7.5 (4.3, 9.6) 0.002 Semen analysis result day 3 FSH Normal 157.0 75.5 Abnormal 51.0 24.5 Female partner AFC status categorized ≥5 140 (95.2%) 44 (83.0%) 0.005 Male partner TESE done <5 7 (4.8%) 9 (17.0%) Yes 27.0 13.0 Female partner day 5.3 (3.5, 7.7) 5.8 (4.3, 10.0) 0.230 No 181.0 87.0 3 LH status, median __________________________________________ (IQR) Female partner day 122.4 (62.0, 243.0) 93.0 (50.4, 150.0) 0.089 3 estradiol, median (IQR) _________________________________________________________ Clinical pregnancy rate and predicting factors **p-values are calculated based on Mann-Whitney U test for continuous independent variables and Fisher’s exact test for categorical independent The long protocol was used for ovarian variables stimulation in 136(68.3%) of the cases. The overall pregnancy rate was found to be 30.1% (60/199). Among these, 5(25%) of them were already delivered, 21(35%) aborted and embryo transfer, and day 5 embryo transfer with a positive 24(40%) of the pregnancy were ongoing at the pregnancy test. However, there were no statistically time of data collection. In bivariate analysis, significant associations between the pregnancy test result significant associations were observed between and BMI, Day 3 FSH, and AMH (Table 3). the female partner age, antral follicular count (AFC), number of oocytes retrieved (good responders), one embryo transfer, grade one 4 Ethiopian Journal of Reproductive Health (EJRH) July, 2021 Volume 14, No. 2 Table 3. Bivariate analysis of independent variables by the pregnancy test result __________________________________________________________________________________________________ Characteristics Negative (n=139) Positive (n=60) p-value* __________________________________________________________________________________________________ Age of female partner, mean (SD)33.7 (4.5) 31.3 (4.4) <0.001 Age of female partner <35 years 74 (53.2%) 44 (74.6%) 0.007 ≥35 years 65 (46.8%) 15 (25.4%) Female partner body mass index <18.3 4 (2.9%) 2 (3.3%) 0.30 18.3-24.9 86 (61.9%) 44 (73.3%) 25-29.9 29 (20.9%) 11 (18.3%) 30-34.9 17 (12.2%) 2 (3.3%) >34.9 3 (2.2%) 1 (1.7%) Age of male partner, mean (SD) 40.4 (6.4) 37.3 (5.2) 0.071 Male partner TESE done Yes 19 (13.7%) 7 (11.7%) 0.82 No 120 (86.3%) 53 (88.3%) Female partner day 3 FSH status, median (IQR) 6.0 (4.3, 7.8) 5.0 (3.5, 7.0) 0.060 Female partner day 3 LH status, median (IQR) 5.5 (3.8, 9.1) 5.5 (3.6, 7.1) 0.78 Female partner day 3 estradiol, median (IQR) 98.0 (51.6, 230.0) 150.0 (86.0, 279.0) 0.082 Female partner AFC status, median (IQR) 10.0 (6.0, 12.0) 12.0 (10.0, 15.0) <0.001 Female partner AFC status categorized ≥5 121 (90.3%) 56 (98.2%) 0.001 <5 13 (9.7%) 1 (1.8%) Female partner AMH serostatus, median (IQR) 0.7 (0.4, 1.2) 1.4 (0.6, 7.1) 0.061 Number of oocytes retrieved, median (IQR) 5.0 (3.0, 10.0) 10.0 (6.0, 13.0) <0.001 Good responders 91 (65.5%) 56 (93.3%) <0.001 Poor responders 48 (34.5%) 4 (6.7%) Number of embryos transferred 1 37 (26.8%) 4 (6.7%) 0.001 2 79 (57.2%) 49 (81.7%) 3 22 (15.9%) 7 (11.7%) Type of protocol used Long protocol 85 (62.5%) 51 (86.4%) 0.063 Short protocol 19 (14.0%) 2 (3.4%) Minimal stimulation 32 (23.5%) 6 (10.2%) Day of embryo transfer Day 5 68 (48.9%) 50 (83.3%) <0.001 Day 3 71 (51.1%) 1 Grades of embryos transferred 1 99 (71.2%) 56 (93.3%) 0.004 2 32 (23.0%) 4 (6.7%) 3 5 (3.6%) 0 (0.0%) Semen analysis result Normal 103 (74.1%) 47 (78.3%) 0.59 Abnormal 36 (25.9%) 13 (21.7%) __________________________________________________________________________________________ *p-values are calculated based on independent t-test or Mann-Whitney U test for continuous independent variables and Fisher’s exact test for categorical independent variables 5  Ethiopian Journal of Reproductive Health (EJRH) July, 2021 Volume 14, No. 2 Multivariable regression of factors associated with day 3 FSH, and AFC count ≥5 were significantly the pregnancy test result associated with good ovarian stimulation response. Multiple logistic regression was run using variables However, given the very brief history of IVF in associate with the pregnancy on bivariate analysis Ethiopia, much is to be done to contextualize the to control confounders. A full model assessing the various nuisances in the field. relationship between the pregnancy test result and predictor variables was constructed after which non- One unique challenge is knowing the age of women significant variables were removed by a backward seeking the service. Although age is one of the procedure using the likelihood ratio test (p<0.05). most important predictors of ovarian response, its Likelihood ratio test (lrtest) was significant when utility in our setting remains questionable. Most variables number and grade of the embryo were of our clients do not know their exact age and it removed by the backward procedure (see Table 4). is usually hard for the physician to ascertain the The odds of getting pregnant is 61% less among stated age as most women have no birth certificate. participants with female partners age ≥35 years, As described above, close to one-third (64.5%) of AOR 0.39, CI 0.18-0.83 with a p-value of 0.015. the study participants did not know their exact date Clients with Day 5 embryo transfer were 3.28 times of birth. In the literature, age has been consistently more likely to get pregnant compared to those with shown to be one of the most important predictors day 3 embryo transfer, p-value=0.006, CI 1.42- of IVF success and fertility in general 3-5. Our study 7.62(see Table 4). also depicted the reported age had a significant association with ovarian response and a positive Table 4: Multiple logistic regression model pregnancy test. This underlines the importance of _________________________________________________ age, even in setups where the exact age cannot be Pregnancy test AOR P>z 95% Confidence Interval ascertained. _________________________________________________ Age In the current study day, 3 FSH was associated with <35 a good response to ovarian stimulation. This is ≥35 years 0.39 0.015 0.18 0.83 AFC categorized in line with many types of research done over the ≥5 years. Muasher and collaborators6 reported that the <5 0.34 0.326 0.04 2.94 measurement of serum levels of FSH, LH, and E2 Oocytes retrieved on day 3 of the basal menstrual cycle was a predictor Good responders of controlled ovarian hyperstimulation (COH) Poor responders 0.35 0.083 0.11 1.15 response and IVF outcome. Subsequent studies Embryo transfer day ascertained the clinical significance of defined Day 3 Day 5 3.28 0.006 1.42 7.62 thresholds for such hormones in addition to their _________________________________________________ relationship to the woman’s age, thus further defining the concept of ovarian reserve5,9-11. It was reported that the combined use of age and basal DISCUSSION FSH in counseling patients improved the accuracy Good responders ((≥4 oocytes retrieved) accounts of prognosis and provide an index of functional for 76.4% of the cases in the current study. The ovarian reserve11. IVF pregnancy rate at the center for reproductive Anti-Mullerian hormone (AMH), and antral medicine (CFRM, SPHMMC) was 30.1%. Good follicular count (AFC) were also being used for responders ((≥4 oocytes retrieved) accounts for predicting ovarian response (18, 19). Recently, 152(76.4%) of the cases. Age of female partner, much attention has been given to the measurement 6 Ethiopian Journal of Reproductive Health (EJRH) July, 2021 Volume 14, No. 2 of AMH20. AMH is produced solely by the were not associated with pregnancy test result in granulosa cells of growing pre-antral and small our study. These tests are better at predicting the antral ovarian follicles and shows little inter-and ovarian response than the pregnancy outcome and intra-cycle variability. AMH is an accurate predictor our study was not adequately powered to detect the of excessive response to ovarian hyperstimulation relationship between these laboratory investigations 16,18. However, our study failed to show a and pregnancy outcome. significant association. This can be explained by The current study is the first of its kind in Ethiopia the small number of patients that had AMH tests and will contribute to the very few publications from done. Besides, since the investigation is expensive, the whole continent. The study explored possibly we did it for those with diminished ovarian reserve clinical parameters to predict ovarian response and to ascertain the assessment determined by antral pregnancy outcome in a resource-limiting setting. follicle count examination. Thus, the AMH was This will help reproductive endocrinologists almost exclusively done on possible poor responders to identify which parameters to use for clinical which were unlikely to have a good IVF outcome. decisions. However, in the current study, we did The current study showed AFC ≥5 was significantly not do multiple logistic to control confounders for associated with a good ovarian response which is outcome ovarian response because of missing values congruent with most studies16, 18, 19. on fully model regression. Furthermore, because In the current study, multiple regression analysis of the retrospective nature of the study, important showed that the reported age of female partner <35 variables like some demographic information, years and day 5 embryo transfer were associated causes, and duration of infertility were difficult to with positive clinical pregnancy rate. Female clients retrieve from the electronic record and were not with age less than 35 were four times more likely included in the analysis. to get pregnant. This reflects ovarian function and underlying ovarian response which is associated CONCLUSION with age less than 35 in the current study. Moreover, Even though nearly two-third of the study the finding was in line with a systematic review and participants did not know their exact date of individual studies which showed young age to be birth, the reported age of female partner <35 years associated with pregnancy occurrence22-24. The is associated with both good ovarian response current study showed day 5 transfer to be more and occurrence of pregnancy emphasizing its than three times more likely to result in pregnancy. importance for clinical decision making. Day 3 However, many studies showed there is no difference FSH and AFC ≥5 were associated with good ovarian in pregnancy rate between day 3 transfer and day 5 stimulation response. Therefore, we recommend the transfer, with fewer cycles with no transfer due to combination of female partner age, day 3 FSH, and very poor-quality embryos or arrested development AFC ≥5 to predict ovarian response in low resource when pushing today 5 25,26. This might be because settings, since variables can be readily available of the practice in our setting in which only patients without much cost to patients. Furthermore, we with less number and quality of embryo undergo would like to recommend follow-up studies with a day 3 transfer compared to routine day 5 embryo larger sample size and prospective cohort design to transfer for those with good number and grade of appropriately compare the different predictors of the embryo resulting in more pregnancies. Cycle ovarian response in our setting to develop evidence- day 3 serum FSH, LH, and E2 levels, measurement based set up specific IVF protocols and guidelines. of AMH, and the estimation of the basal AFC 7  Ethiopian Journal of Reproductive Health (EJRH) July, 2021 Volume 14, No. 2 Abbreviations AFC: Antral Follicular Count AMH: Anti-Mullerian Hormone BMI: Body Mass Index CCCT: Clomiphene Citrate Challenge test CFRM: Center for Reproductive Medicine COH: Controlled Ovarian Hyperstimulation E2: Estradiol FSH: Follicle Stimulating Hormone FMOH: Federal Ministry of Health. IVF: In Vitro Fertilization LH: Luteinizing Hormone SPHMMC: St. Paul’s Hospital Millennium Medical College Funding. The authors received no specific funding for this work. Competing Interests. The authors declare that they have no competing interests. ACKNOWLEDGMENTS We would like to thank the institutional review board of SPHMMC for the ethical clearance and the hospital administration for permission to conduct the study. Furthermore, we would like to thank all study participants and data collectors for their contribution to the success of this work. CORRESPONDING AUTHOR: Lemi Belay St. Paul’s Hospital Millennium Medical College, Addis Ababa, Ethiopia Email: [email protected] 8 Ethiopian Journal of Reproductive Health (EJRH) July, 2021 Volume 14, No. 2 REFERENCES 1. The global prevalence of infertility, infecundity, and childlessness, analysis by WHO available from https://www.who.int/ reproductivehealth/topics/infertility/burden/en/ 2. Deribe, Anberbir. 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