Undetectable Basal Ovarian Stromal Blood Flow in Infertile Women is Related to Low Ovarian Reserve
By Younis, Johnny S Haddad, Sami; Matilsky, Moshe; Radin, Orit; Ben- Ami, Moshe
Abstract To gain insight into the physiological significance of basal ovarian stromal blood flow and to assess whether its detection ability is related to ovarian reserve in infertility patients undergoing in vitro fertilization (IVF)-embryo transfer (ET) treatment. Thirty two consecutive infertile women scheduled for IVF- ET treatment were prospectively evaluated. Basal ovarian hormonal, ovarian volume and stromal blood flow studies were performed on day 3 of a natural cycle before treatment. The performer of the ultrasound studies was blinded to the clinical data. Women in the study were divided into two groups in accordance with estradiol level on the day of administration of human chorionic gonadotropin. Day-3 follicle-stimulating hormone and ovarian volume were significantly poorer in the women with low (group A) as compared with good (group B) ovarian reserve. Likewise, the numbers of follicles >/=14 mm in diameter, oocytes retrieved and embryos achieved were significantly lower in group A than group B. Six clinical pregnancies were obtained in group B, whereas no pregnancy was obtained in group A. Nine out of the 15 (60%) women in group A had undetectable basal stromal blood flow in at least one of the ovaries, whereas only one of the 17 (6%) women in group B had undetectable flow (p
Keywords: Ovarian stroma, blood flow, ovarian reserve, in vitro fertilization-embryo transfer, Doppler
The mass of primordial follicles in the human female is considered the biomarker of ovarian age [I]. Indeed, quantitative histological studies of human ovaries have confirmed that the total number of follicles declines through out life, and that very few, if any, remain shortly after menopause . The physiological decrease of primordial follicles in the human ovary in conjunction with the decline in oocyte quality is referred to as low ovarian reserve . Although the occurrence of this event is age-related, its onset in women is highly variable and its duration before ovarian senescence is usually unpredictable. It is estimated that the number of primordial follicles at menarche is about 300 000 and that it drops to only a few hundreds or thousands at the end of reproductive life . Moreover, it is estimated that there is an accelerated loss of primordial follicles beginning at the age of 37-38 years, when a critical number of about 25 000 follicles is reached [1,2]. This typically precedes the menopause by 10-12 years. However, there is still a large, distinct variation between women, making it difficult to predict individual reproductive performance. Our understanding of the mechanisms that control ovarian reserve is still developing. One explanation could be related to life in utero; that is, the timing of oogonial stem cell elimination before birth could designate the size of the primordial follicle store in the ovary and therefore determine an upper limit on reproductive life span . However, other mechanisms could also be involved.
In the current era of assisted reproduction and with die introduction of intracytoplasmic sperm injection to treat severe male-factor infertility, ovarian reserve has turned out to be the major determinant of reproductive performance. Therefore, it has become exceptionally important to evaluate the ovarian reserve before counseling for such treatments. Indeed, several studies in the last decade have focused on the ability to assess ovarian reserve as a means of predicting assisted reproduction potential. Endocrine studies proposed for examining ovarian reserve can be divided into static and dynamic tests . Static studies generally are agreed to be undertaken on day 3 of a natural cycle and commonly are termed ‘basal tests’. High basal levels of follicle-stimulating hormone (FSH)  and estradiol (E2) , in addition to low inhibin B , are usually considered to be related to low ovarian reserve. Dynamic studies have been performed to assess ovarian reserve after challenge with clomiphene citrate , gonadotropins  or gonadotropin-releasing hormone (GnRH) agonist . However, it seems that these tests do not have absolute sensitivity and specificity . Moreover, hormonal determinations may have inter- cycle variability , are laborious and expensive, and have not achieved wide clinical acceptance. Therefore, the search for more accurate, simple and agreeable parameters of ovarian reserve is still ongoing.
The combination of transvaginal ultrasound and pulsed color Doppler is increasingly used in gynecology to assess the hemodynamic changes in various physiological and pathological situations of the pelvic organs [14,15]. Moreover, in the last few years, some investigators have examined the feasibility of employing this technique in the uterine as well as the intraovarian circulations, in order to predict the outcome of in vitro fertilization (IVF)- embryo transfer (ET) treatment [16-26]. In addition, more recentiy three-dimensional power Doppler technology has been employed within the ovarian stroma to investigate its relationship with IVF-ET outcome [27,28] .
Employing two-dimensional color Doppler and addressing the intraovarian vasculature, studies have evaluated blood flow following pituitary downregulation [19,20], during superovulation [21-23] and oocyte pick-up [21-24], or through the luteal phase [21- 23]. Other studies have evaluated stromal blood flow in the early follicular phase [21,25,26], specifically in a natural cycle and before administering any hormonal therapy. However, the results of the previously published reports are still conflicting  . Moreover, to the best of our knowledge, the issue of detection or not of the basal stomal ovarian blood flows in infertile women and its relationship with ovarian reserve has not yet been investigated in a targeted study.
Therefore we undertook the present study in order to evaluate, in a prospective fashion, the physiological significance of basal ovarian stromal blood flow employing two-dimensional transvaginal pulsed Doppler and color Doppler technologies. Specifically, we wished to assess whether detection or non-detection of basal stromal flow is related to ovarian reserve in infertility patients undergoing rVF-ET treatment.
Thirty-two consecutive women attending the Poriya Reproductive Medicine Unit in Tiberias, Israel, and scheduled for IVF-ET treatment, were prospectively evaluated in this preliminary work. AU women were spontaneously menstruating and had no clinical signs of menopause. Both ovaries were present in all women. Women with poor visualization of the ovaries (e.g. abdominal position) or with ovarian cyst >25 mm were excluded from the study. Infertile women eligible for IVF with day-3 FSH >10 IU/1 but still with regular menstruation were not excluded from this study. The etiologies of infertility in these patients were mechanical factor in six, male factor in eight, ovulatory dysfunction in six, unexplained infertility in ten and endometriosis in two cases. AU of the women were determined to have normal uterine cavities by hysterosalpingography and/or hysteroscopy. The results of the ovarian flow studies did not affect the course of IVF treatment in any patient.
Basal ovarian reserve studies including serum FSH and E2 as well as luteinizing hormone (LH) levels were obtained on day 3 of a natural cycle, one month prior to initiating IVF-ET treatment and following at least 3 months of no hormonal therapy.
On the same day as blood sampling, ovarian volume and basal stromal ovarian blood flow studies were performed in all women. Basal ultrasound studies were performed by one performer (S.H.) blinded to the clinical and endocrinological data as well as previous infertility treatment results. Ovarian volume and blood flow scans were performed employing a two-dimensional endovaginal probe with pulsed Doppler and color Doppler facilities (Acuson 128- P-10; Acuson, Mountain View, CA, USA). Ovarian volumes were calculated as the volume of an ellipsoid, i.e. length ? width ? depth ? p/6. The total basal volume of both ovaries was evaluated in each patient. The ultrasound frequency for B-mode, pulsed Doppler and color Doppler was 7 MHz and the high-pass filter was set on 100 Hz. Intraovarian blood flow was assessed in each ovary by examining blood vessels in the ovarian stroma (i.e. any small artery in the ovarian stroma not close to the surface of the ovary or near the wall of a follicle). Areas of maximum color intensity, representing the greatest Doppler frequency shifts, were selected for pulsed Doppler examination. Peak systolic blood flow velocity waveforms were dius detected and optimal flow velocity waveforms were selected for analysis. A recording was considered satisfactory for measurement when there were at least three equally intense waveforms in a row (Figure 1). An ovary that did not show waveforms for accurate analysis was concluded as an ovary with no detectable flow. The pulsatility index (PI), resistance index (RI), and peak systolic blood flow velocity were measured. These values were calculated electronically from smooth curves fitted to the maximum envelope of good-quality waveforms over three cardiac cycles. The formulas used to calculate the angle-independent PI and RI were: PI = (S-D)ZA and BI = (S-D)IS, respectively, where S is the maximum, D the end- diastolic and A the mean maximum Doppler shift frequency throughout the cardiac cycle.
The long protocol, starting on day 21 of the cycle, with GnRH agonist (GnRH-a) for IVF-ET, was similarly employed in each patient. Downregulation was achieved after intramuscular administration of GnRH-a (Decapeptyl CR(R) 3.75 mg; Ferring, Malmo, Sweden) and was assured by serum E2 levels 1468 pmol/1. Transvaginal oocyte retrieval was performed 34-36 h after hCG administration under ultrasound guidance. The treatments of oocytes, sperm and embryos, as well as the ET technique, were performed as described previously . Luteal support was administered in all patients employing intramuscular injection of progesterone (P) in oil (Gestone(R) Paines and Byrne Limited, Greenford, UK), 50 mg/day.
Figure 1 . Normal color Doppler image of the basal stromal blood flow in a natural cycle. Note that areas of maximum color intensity within the ovary, representing the greatest Doppler frequency shifts, are selected for pulsed Doppler examination. Please see colour online.
Women in the study were divided into two groups in accordance with E^sub 2^ level on hCG administration day. Group A included women with E2 level 5505 pmol/ 1. Ovarian reserve parameters, IVFET results and basal ovarian stromal flow data were compared between the two groups.
Sera obtained for basal FSH and LH measurements were analyzed by microparticle enzyme immunoassay (IMx; Abbott, Abbott Park, IL, USA). The intra-assay and inter-assay coefficients of variation were
Data were analyzed using Student’s t test, the X2 test and the Mann-Whitney two sample test (unpaired, non-parametric), wherever appropriate. Significance was interpreted as p
Of the 32 women participating in the study, 15 were included in group A (low reserve) and 17 in group B (good reserve). Patients’ characteristics, including age, body mass index, duration and degree of infertility, were similar between group A and group B of the study (Table I).
Day-3 FSH level was significantiy higher in group A than group B: 10.3 +- 4.3 vs. 6.9 +- 1.5 IU/1, respectively. Moreover, total ovarian volume was significantiy lower in group A than group B, corresponding to 9.1+-4.7 and 18.2 +- 8.2 cm^sup 3^, respectively. Day-3 E2 and LH levels were similar in both groups (Table II).
As anticipated from the inclusion criteria of this study, the mean E2 level on hCG day was significantly higher in group B compared with group A: 9586 +- 3975 vs. 3197 +- 1380 pmol/1, respectively. Moreover, as expected, the number of >/=14 mm follicles on hCG day, number of oocytes retrieved and number of embryos achieved were significantly higher in group B than group A, being 12.8 + 4.9, 15.7 +- 5.5 and 8.4 +- 6.9 in group B and 6.2 +- 3.9, 4.0 +- 3.2 and 2.7 +- 3.4 in group A, respectively (Table II). Three patients in group A and none in group B had no oocytes retrieved on the pick-up day, despite the ultrasound-guided aspiration of more than two follicles in all women. Moreover, the hMG dosage required to achieve stimulation was significantly lower in group B compared with group A: 39.3 + 14.1 vs. 51.1 + 19.4 ampoules, respectively (all p
Table I. Patients’ characteristics in groups A and B.
Table II. Basal hormonal ovarian reserve and ovarian volume studies, and clinical data from in vitro fertilization treatment, in groups A and B.
Table III. Basal ovarian stromal blood flow characteristics in groups A and B.
Six clinical pregnancies were achieved in group B (clinical pregnancy rate 35%); however no pregnancy occurred in group A during the study period.
Twenty-two of the 32 (69%) women in the study had detectable stromal ovarian blood flow in both ovaries on day 3 of a natural cycle, while nine (28%) women had detectable flow in only one ovary. One woman (3%) had no detectable flow in both ovaries. Nine out of the 15 (60%) women in group A had no detectable stromal blood flow on day 3 of the cycle in at least one of die ovaries, whereas this was the case for only one of the 17 (6%) women in group B (Table III). This difference was statistically significant (p
The mean values of peak systolic blood flow velocity, PI and RI did not differ significantly between groups A and B (Table III). Moreover, these values did not differ between right and left ovaries in both groups.
Our results indicate that undetectable ovarian stromal blood flow, on day 3 of a natural cycle, is related to low ovarian reserve in infertile women undergoing IVF-ET treatment. In 60% of women with low reserve, as compared with only 6% (p /=14 mm follicles (on hCG day), greater numbers of oocytes retrieved and embryos achieved, and also a substantially lower hMG requirement. The fact that the pregnancy rate was significandy higher in group B than group A reinforces these findings.
Moreover, the finding that all three women with no oocytes during retrieval (despite follicular development and adequate E2 elevation) came from the low ovarian reserve group further strengthens our conclusion. Our group has recendy presented a study showing that complete absence of oocytes during retrieval is a manifestation of low ovarian reserve .
It could be argued that undetectable ovarian stromal blood flow could also be the result of bias or a technical problem related to the performance of the transvaginal ultrasound and/or the color Doppler. However, since our study was prospectively designed, flow studies were performed before basal hormonal studies results were received and actual IVF-ET treatment concluded. Therefore, the clustering of women with no detectable stromal flow in the low ovarian reserve group seems to be real. Moreover, the fact that die performer of the flow studies was blinded to all clinical data strengthens our conclusion. Furthermore, these arguments support the assumption that undetectable basal stromal blood flow is not solely a technical issue, but rather is linked to the pathophysiology of low ovarian reserve.
It is currently well accepted that the follicle destined to ovulate is recruited with many odier primordial follicles, in the early follicular phase. Some of these follicles will be committed to growth, developing to preantral and antral follicles; however only one follicle (in most cases of natural cycles) will be selected to ovulate. All other follicles will become atretic. Although the mechanism responsible for reinitiating follicular recruitment remains an enigma, it is acceptable to assume that it is not purely an FSH-dependent action, but local regulators, mainly growth factors, chiefly mediate it  . Moreover, it is believed that die number of follicles that are recruited in each cycle depends upon the size of the residual pool of inactive primordial follicles , i.e. ovarian reserve. It is our belief that the larger is the ovarian reserve, the more the stromal vasculature is developed. Therefore, women with good ovarian reserve will show optimal blood flow studies while women with low reserve will not.
Our results are conceptually in agreement with other published papers reporting an association between ovarian volume [31,32], and number of small follicles , before gonadotropin administration, with ovarian responsiveness during IVF-ET. Syrop and colleagues  in a retrospective study and Lass and associates  in a prospective study suggested that ovarian volume of
Several studies have examined the feasibility of using ovarian flow studies before gonadotropin stimulation [19,20,25,26] to assess ovarian response during IVF-ET treatment. Although some studies did find a correlation between flow studies and ovarian response [19,25,26], others did not . Several explanations could clarify the differences in the results. The first is related to the timing of performing the flow studies. Some of these studies performed the flow procedure following GnRH-a downregulation [19,20], while others determined the flow in die early follicular phase in a natural cycle before starting any hormonal treatment [25,26] . The second is linked to patient selection criteria. Some studies have excluded infertile women with day-3 FSH >10 IU/1 [19,20] or >12.5 IU/1 , and one excluded women >40 years of age  . The third may be connected to the methodology of performing the flow studies. While some have used the pulsed and color Doppler facilities [19,20,25], others have used only the power Doppler technology employing a semi- quantitative score .
Our study was performed in a prospective manner during a natural cycle, one month preceding IVF-ET treatment. Patients older than 40 years and women with day-3 FSH >10 IU/1 were not excluded. Women included were eligible for IVF-ET treatment, had spontaneous menstruation, and were capable of achieving at least two mature follicles and E2 >1468 pmol/l on hCG day. Most importantly, the issue of detection or not of stromal ovarian flow was specifically looked for in our study. Basal stromal peak systolic blood flow velocity, PI and RI did not differ between the patients with low and good ovarian reserve in our study. However, flow detection was the only significant parameter that differed between the low and good ovarian reserve infertile women.
Women with low ovarian reserve are noted to have spontaneous ovulation. However, in each cycle the primary cohort of recruited follicles seems to get smaller and smaller. It is possible that, under these circumstances, only the ovary that is selected to have the dominant follicle will show stromal activity and therefore will have stromal blood flow that could be detected by pulsed color Doppler. This could explain why nine out of ten women who did not show basal stromal blood flow in our study had no flow in only one ovary. This speculation needs to be further explored by future targeted studies.
In conclusion, this preliminary work suggests that basal ovarian stromal blood flow is related to ovarian reserve in infertile women undergoing IVF-ET treatment. Undetectable basal ovarian stromal blood flow, in at least one of the ovaries, is associated with low ovarian reserve. The fact that the flow studies were performed blinded to the clinical data strengthens the assumption that undetectable basal stromal blood flow is not solely a technical issue, but related to the physiology of ovarian aging.
1. Faddy MJ, Gosden RG A mathematical model of follicle dynamics in the human. Hum Reprod 1995;10:770-775.
2. Richardson SJ, Senikas V, Nelson JF. Follicular depletion during the menopausal transition: evidence for accelerated loss and ultimate exhaustion. J Clin Endocrinol Metab 1987;65:1231-1237.
3. Scott RT, Hofmann GE. Prognostic assessment of ovarian reserve. Fertil Steril 1995;63:1-11.
4. Speroff L, Glass RH, Kase NG. Clinical gynecologic endocrinology and infertility. 5th ed. Baltimore (MD): Williams & Wilkins; 1994. pp 183-230.
5. Faddy MJ, Gosden RG. A model conforming the decline in follicle numbers to the age of menopause in women. Hum Reprod 1996;11:1484-1486.
6. Younis JS, Haddad S, Matilsky M, Ben-Ami M. Premature luteinization: could it be an early manifestation of low ovarian reserve? Fertil Steril 1998;69:461-465.
7. Muasher SJ, Oehninger S, Simonetti S, Matta J, Ellis LM, Liu H- C, Jones GS, Rosenwaks Z. The value of basal and/or stimulated serum gonadotropin levels in prediction of stimulation response and in vitro fertilization outcome. Fertil Steril 1988;50:298-307.
8. Licciardi FL, Liu H-C, Rosenwaks Z. Day 3 estradiol serum concentrations as prognosticators of ovarian stimulation response and pregnancy outcome in patients undergoing in vitro fertilization. Fertil Steril 1995;64:991-994.
9. Seifer DB, Lambert-Messerlian G, Hogan JW, Gardiner AC, Blazar AS, Berk CA. Day 3 serum inhibin-B is predictive of assisted reproductive technologies outcome. Fertil Steril 1997;67:110-114.
10. Navot D, Rosenwaks Z, Margalioth EJ. Prognostic assessment of female fecundity. Lancet 1987;2:645-647.
11. Fanchin R, de Ziegler D, Olivennes F, Taieb J, Dzik A, Frydman R. Exogenous follicle stimulating hormone ovarian reserve test (EFORT): a simple and reliable test for detecting ‘poor responders’ in m-OTtro-fertilization. Hum Reprod 1994;9:1607-1611.
12. Padilla SL, Bayati J, Garcia JE. Prognostic value of the early serum estradiol response to leuprolide acetate in in vitro fertilization. Fertil Steril 1990;53:288-294.
13. Scott RT Jr, Hofmann GE, Oehninger S, Muasher SJ. Intercycle variability of day 3 follicle-stimulating hormone levels and its effect on stimulation quality in in vitro fertilization. Fertil Steril 1990;54:297-302.
14. Kurjak A, Zalud I, Jurkovic D, Alfirevic Z, Miljan M. Transvaginal color Doppler for the assessment of the pelvic circulation. Acta Obstet Gynecol Scand 1989;68:131-135.
15. Campbell S, Bourne TH, Waterstone J, Reynolds K, Crayford TJB, Jurkovic D, Okokon EV, Collins WP. Transvaginal color blood flow imaging of the peri-ovulatory follicle. Fertil Steril 1993;60:433-438.
16. Steer CV, Campbell S, Tan SL Crayford T, Mills C, Mason BA, Collins WP. The use of transvaginal color flow imaging after in vitro fertilization to identify optimum uterine conditions before embryo transfer. Fertil Steril 1992; 57:372-376.
17. Favre R, Bettahar K, Grange G, OhI J, Arbogast E, Moreau L, Dellenbach P. Predictive value of transvaginal uterine Doppler assessment in an in-vitro fertilization program. Ultrasound Obstet Gynecol 1993;3:350-353.
18. Weiner Z, Thaler I, Levron J, Lewit N, Itskovitz-Eldor J. Assessment of ovarian and uterine blood flow by transvaginal color Doppler in ovarian-stimulated women: correlation with the number of follicles and steroid hormone levels. Fertil Steril 1993;59:743- 749.
19. Engmann L, Sladkevicius P, Agrawal R, Bekir JS, Campbell S, Tan SL. Value of ovarian stromal blood flow velocity measurement after pituitary suppression in the prediction of ovarian responsiveness and outcome of in vitro fertilization treatment. Fertil Steril 1999;71:22-29.
20. Ng EHY, Tang OS, Chan CCW, Ho PC. Ovarian stromal blood flow in the prediction of ovarian response during in vitro fertilization treatment. Hum Reprod 2005;20:3147-3151.
2 1 . Tekay A, Martikainen H, Jouppila P. Blood flow changes in uterine and ovarian vasculature, and predictive value of transvaginal pulsed color Doppler ultrasonography in an in-vitro fertilization program. Hum Reprod 1995;10:688-693.
22. Lunenfeld E, Schwartz I, Meizner I, Potashnik G, Glezerman M. Intraovarian blood flow during spontaneous and stimulated cycles. Hum Reprod 1996;11:2481-2483.
23. Bassil S, Wyns C, Toussaint-Demylle D, Nisolle M, Gordts S, Donnez J. The relationship between ovarian vascularity and the duration of stimulated in in-vitro fertilization. Hum Reprod 1997;12:1240-1245.
24. Chui DKC, Pugh ND, Walker SM, Gregory L, Shaw RW. Follicular vascularity – the predictive value of transvaginal power Doppler ultrasonography in an in-oitro fertilization program: a preliminary report. Hum Reprod 1997;12:191-196.
25. Zaidi J, Barber J, Kyei-Mensah A, Bekir J, Campbell S, Tan SL. Relationship of ovarian stromal blood flow at baseline ultrasound scan to subsequent follicular response in an in vitro fertilization program. Obstet Gynecol 1996;88:779-784.
26. Popovic-Todorovic B, Loft A, Lindhard A, Bangsboll S, Andersson AM, Andersen AN. A prospective study of predictive factors of ovarian response in ‘standard’ IVF/ICSI patients treated with recombinant FSH. A suggestion for a recombinant FSH dosage normogram. Hum Reprod 2003; 18:781-787.
27. Jarvela IY, Sladkevicius P, Kelly S, Ojha K, Campbell S, Nargund G. Quantification of ovarian power Doppler signal with tree- dimensional ultrasonography to predict response during in vitro fertilization. Obstet Gynecol 2003;102:816-822.
28. Pan HA, Wu MH, Cheng YC, Wu LH, Chang FM. Quantification of ovarian stromal Doppler signals in poor responders undergoing in vitro fertilization with three-dimensional power Doppler ultrasonography. Am J Obstet Gynecol 2004;190:338-344.
29. Laufer N, Grunfeld L, Garrisi GJ. In-vitro fertilization. In: Seibel MM, editor. Infertility – a comprehensive text. Norwalk (CT): Appleton and Lange; 1990. pp 481-511.
30. Younis JS, Skournik A, Radin O, Haddad S, Bar-Ami S, BenAmi M. Poor oocyte retrieval is a manifestation of low ovarian reserve. Fertil Steril 2005;83:504-507.
31. Syrop CH, Wilhoite A, Van-Voorhis BJ. Ovarian volume: a novel outcome predictor for assisted reproduction. Fertil Steril 1995;64:1167-1171.
32. Lass A, Skull J, McVeigh E, Margara R, Winston R. Measurement of ovarian volume by transvaginal sonography before ovulation induction with human menopausal gonadotrophins for in-vitro fertilization can predict poor response. Hum Reprod 1997;12:294- 297. 33. Tomas C, Nuojua-Huttunen S, Martikainen H. Pretreatment transvaginal ultrasound examination predicts ovarian responsiveness to gonadotrophins in in-vitro fertilization. Hum Reproduc 1997;12:220-223.
JOHNNY S. YOUNIS, SAMI HADDAD, MOSHE MATILSKY, ORIT RADIN, & MOSHE BEN-AMI
Reproductive Medicine Unit, Department of Obstetrics and Gynecology, Poriya Medical Center, Tiberias, Israel, affiliated with the Bruce Rappaport Faculty of Medicine, Technion – Israel Institute of Technology, Haifa, Israel
(Received 14 November 2006; revised 26 February 2007; accepted 5 March 2007)
Correspondence: J. S. Younis, Reproductive Medicine Unit, Department of Obstetrics and Gynecology, Poriya Medical Center, Tiberias 1 5208, Israel. Tel: 972 4 6652490. Fax: 972 4 6080405. E- mail: email@example.com
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