European Journal of Obstetrics & Gynecology and Reproductive Biology 166 (2013) 65–69

The association between poor ovarian response and thrombophilia inassisted reproduction

Johnny S. Younis a,b,*, Moshe Ben-Ami a,b, Ido Izhaki c, Jimmy Jadaon a, Benjamin Brenner d,e, Galit Sarig d,e

a Reproductive Medicine Unit, Department of Obstetrics & Gynecology, Poriya Medical Center, Tiberias, Israelb Faculty of Medicine, Bar-Ilan University, Galilee, Israelc Department of Evolutionary and Environmental Biology, University of Haifa, Haifa, Israeld Thrombosis and Hemostasis Unit, Rambam Health Care Campus, Haifa, Israele Rappaport Faculty of Medicine, The Technion-Israel Institute of Technology, Haifa, Israel

A R T I C L E I N F O

Article history:

Received 20 January 2012

Received in revised form 26 August 2012

Accepted 21 September 2012

Keywords:

Assisted reproductive technologies

IVF-ET

Poor ovarian response

Low ovarian reserve

Ovarian aging and thrombophilia

A B S T R A C T

Objective: To investigate the association between thrombophilic risk factors and poor ovarian response

in an assisted reproductive technologies (ART) setting.

Study design: This is a preliminary prospective cohort study in a university affiliated reproductive

medicine unit. Eighty-nine infertile women undergoing IVF-ET treatment were recruited. Following IVF-

ET treatment, the 28 women that had �3 oocytes on retrieval were the study group, and the 61women

that had � 4 oocytes on retrieval were the control group. All women underwent ovarian reserve testing

and thrombophilia work-up prior to treatment.

Results: Patients’ characteristics, except for chronological age, were similar between the two groups.

Women in the study group had clear manifestations of low ovarian reserve, evident by ovarian reserve

testing, controlled ovarian hyper-stimulation and IVF-ET treatment results, as compared to the control

group. The incidence of all thrombophilias tested was similar between the study and control group.

Moreover, the incidence of any single or combined thrombophilia was also similar between the two

groups. Logistic regression model analysis and Pearson correlation tests did not show significant

correlation between number of oocytes retrieved and thrombophilia. Furthermore, basal ovarian reserve

tests did not differ between women with and without thrombophilia. When only the 48 women � 35

years of were analyzed, the five women in the study group had significantly higher incidence of a single

as well as combined thrombophilia as compared to the 43 controls.

Conclusions: Thrombophilic risk factors have no correlation with poor ovarian response in the general

infertile population undergoing ART. Whether premature low ovarian reserve is linked to thrombophilia

remains to be established.

� 2012 Elsevier Ireland Ltd. All rights reserved.

Contents lists available at SciVerse ScienceDirect

European Journal of Obstetrics & Gynecology andReproductive Biology

jou r nal h o mep ag e: w ww .e lsev ier . co m / loc ate /e jo g rb

1. Introduction

Ovarian aging is a continuous process governed by a gradualdecrease in the quantity and quality of the oocytes starting in-uteroand extending through the menopausal transition. On average,fecundity begins gradually decreasing at age 31 and at about 37–38years of age a significant decline in fertility occurs [1–3].

Nevertheless, there is a wide variability in the ovarian reserveamong women of the same age group, with a distribution range of20 years [4]. It has been well recognized that about 10% of women

* Corresponding author at: Reproductive Medicine Unit, Department of Obstet-

rics & Gynecology, Poriya Medical Center, Tiberias 15208, Israel.

Tel.: +972 40 6652275; fax: +972 4 6080405; mobile: +972 505 286981.

E-mail address: jsy@netvision.net.il (J.S. Younis).

0301-2115/$ – see front matter � 2012 Elsevier Ireland Ltd. All rights reserved.

http://dx.doi.org/10.1016/j.ejogrb.2012.09.015

in the general population reach their menopause by the age of 45years [5–7]. A significant proportion of women of reproductive agewill exhibit follicular apoptosis, depletion of ovarian reserve and asignificant decline in fertility at a much earlier age; considerablybefore the age of 37–38 years, leading to ‘‘premature’’ low ovarianreserve or ovarian aging [3].

Although the exact mechanism behind ovarian aging is not yetclear, it is believed today to be multi-factorial and possibly inter-related to different medical, lifestyle, autoimmune, genetic and asyet unknown factors [3].

Several observations from the last few years might suggest thatlow ovarian reserve could be related to vascular homeostasis andtendency for thrombosis, i.e. thrombophilia. Unexplained infertility,an identified risk factor of ovarian aging [8–10], has been associatedwith thrombophilia [11,12]. Repeat IVF failure, a major feature ofovarian aging [13], has also been related to thrombophilia [14–16].

J.S. Younis et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 166 (2013) 65–6966

Moreover, decreased ovarian stromal blood flow has been associatedwith low ovarian reserve [17,18]. Concomitantly, the presence of atleast one mutant allele of factor V Leiden and apolipoprotein E hasbeen shown to be associated with reduced age at natural menopause[19,20]. As well, higher levels of clotting factor VII have been found tobe associated with early menopause [21,22].

Taken together, these observations might imply that thrombo-philic risk factors could be involved in the development of lowovarian reserve. To the best of our knowledge, ovarian aging hasnot been investigated, in a targeted study, in relation tothrombophilia, in an assisted reproductive technologies (ART)setting. Therefore, our objective was to prospectively evaluate theoccurrence of thrombophilia in infertile women undergoing ARTtreatment and to explore whether low ovarian reserve manifestedby poor ovarian response could be related to thrombophilia.

2. Materials and methods

2.1. Study population and design

Eighty-nine unselected and consecutive infertile womenundergoing IVF-ET treatment were prospectively enrolled intothis study. Only women with tubal and male factors orunexplained infertility were included. All women, between 18and 44 years of age and with a body mass index of 18–34 kg/m2,were regularly menstruating with two intact ovaries and noprevious ovarian surgery. Women in the study were found to havenormal uterine cavities by hysterosalpingography and/or hyster-oscopy. Women with polycystic ovary syndrome or endometriosiswere excluded. Moreover, women with previous thrombo-embolicphenomena, under anti-thrombotic treatment or with knownthrombophilia were excluded from evaluation. Patients withuncontrolled thyroid disease, diabetes mellitus or hyperprolacti-nemia were also excluded from the study. Women enrolled couldparticipate in the study only once.

This was a prospective cohort study. The medical staff in the IVFand endocrine laboratories, as well as the ultrasonographers, wereblinded to the drug regimen and conduct of the study. The researchproject was approved by the Poriya Medical Center InstitutionalReview Board and an informed consent form was signed by eachwoman participating in the study.

Following recruitment all women underwent basal ovarianreserve tests and thrombophilia work-up. Following this investi-gation all women underwent controlled ovarian hyperstimulation(COH) and IVF-ET.

2.2. Ovarian reserve tests

Early follicular basal antral follicle count (AFC, 2–9 mm) andovarian volume evaluation were performed in a natural cycle asdescribed previously [23]. Ovarian ultra-sonography was per-formed employing a trans-vaginal probe (5–9 MHz) (Voluson730 expert; General Electric Medical System, Milwaukee, WI).Blood was drawn on the same day for serum basal FSH, LH, andE2 levels as well as FSH/LH ratio evaluation as previouslydescribed [23].

2.3. Thrombophilia work-up

Blood samples were collected by venipuncture into 3.2%sodium citrate tubes and were centrifuged twice at 2000 � g for15 min. Plasma samples were frozen at �70 � 5 8C. Before testing,plasma aliquots were thawed in a 37 � 0.5 8C water bath for 15 min.All coagulation assays and genetic thrombophilic risk factors havebeen performed as previously described [24]. A complete list of thethrombophilic tests performed in the study appears in Table 4.

2.4. IVF treatment

Controlled ovarian hyper-stimulation was chosen on a case-to-case basis according to standard clinical practice. Conventional IVFand/or intra-cytoplasmic sperm injection (ICSI) were performedaccording to the cause of infertility. A detailed description ofsperm, oocyte and zygote handling as well as embryo transfer andluteal phase supplementation in our unit has been publishedpreviously [25].

2.5. Poor ovarian response definition

Following completion of treatment women enrolled weredivided into two groups in accordance with the number of oocytesretrieved. Women with �3 oocytes were included in the studygroup and women with �4 oocytes in the control group. Patients’characteristics, ovarian reserve tests, COH and IVF-ET results aswell as thrombophilia work-up were compared between the twogroups.

The number of retrieved oocytes following conventionalstimulation is a well accepted criterion of ovarian reserve. Severalstudies have adopted the number of 3 or less oocytes on theretrieval day as a manifestation of low ovarian reserve [26,27], andthis criterion has been recently adopted by the ESHRE consensuson the definition of poor response [28].

2.6. Statistical analysis

We analyzed all data using the Software Package for SocialSciences (SPSS) for windows version 15.0 (SPSS Inc. 2006, Chicago,IL, USA). Descriptive procedure was used to evaluate patients’characteristics and each variable is presented as mean � SD. Mann–Whitney U test, independent two sample Student t-test, Chi-square(x2) for crosstab data, Z test for two proportions, Pearson correlationand logistic regression were used wherever appropriate. Normaldistribution was analyzed prior to statistical tests using Wilk–Shapirotest. P value of <0.05 was considered as statistically significant.

Because the sample size was limited to available data, a post hocpower analysis using observed mean differences and standarddeviations demonstrated that this study had at least 80% power todetect differences of 2.0 in FSH level, of 3.5 in AFC, of 900 IU in totalgonadotropin requirement and of 3 in number of follicles �14 mmon hCG day between the study and the control group (based on thetwo-sample t test for unequal n using a two-sided significance levelof 0.05) and to detect differences of 50% in the prevalence of �2combined thrombophilia in women �35 years old (based on thetwo-proportion Z test for unequal n using a one-sided significancelevel of 0.05).

3. Results

Among the 89 women enrolled in the study, 28 had �3oocytes (study group) and 61 had �4 oocytes (control group)retrieved. Patients’ characteristics, including infertility duration,order and etiology, as well as number of previous IVF cycles, weresimilar between the groups. Lifestyle factors including chronicsmoking rate and body mass index (BMI) were also similarbetween the groups. Gravidity, parity, number of children andprevious abortions were also similar (Table 1). The onlysignificant difference was the mean age of women, correspond-ing to 38.7 � 4.2 and 32.6 � 5.2 years in the study and controlgroups respectively.

Basal ovarian reserve tests showed clear signs of low ovarianreserve in the study group as compared to the control group. Thiswas manifested by significantly higher serum FSH level, lower AFCand ovarian volume (Table 2).

Table 1Patient characteristics’ in the study and control groups. Values are presented as mean � SD. Numbers between brackets represent rates.

Study group (n = 28) Control group (n = 61) P

Age 38.7 � 4.2 32.6 � 5.2 <0.001a

Infertility duration (y) 7.0 � 4.2 6.2 � 4.0 0.45a

BMI (kg/m2) 26.5 � 6.2 27.9 � 6.6 0.38a

Infertility order 0.70b

Primary infertility 11(39.3%) 25(40.1%)

Secondary infertility 11(39.3%) 19(31.1%)

Primary and secondary infertility 6(20.4%) 17(27.8%)

Infertility etiology 0.28b

Male factor 13(46.4%) 32(47.0%)

Mechanical factor 2(7.1%) 10(15.0%)

Unexplained 11(39.3%) 13(16.7%)

Combined 2(7.1%) 6(21.0%)

Number of past IVF cycles 4.3 � 4.0 3.1 � 3.4 0.15a

Chronic smoking 4(14.3%) 15(24.6%) 0.27c

Gravidity 1.3 � 1.6 1.0 � 1.1 0.34a

Parity .7 � 0.80 0.5 � 0.6 0.23a

Number of children .7 � 0.80 0.5 � 0.7 0.37a

Number of abortions 0.6 � 0.9 0.4 � 0.7 0.32a

a Mann–Whitney U test.b Chi Square test for crosstab data.c Z-test for two proportions.

Table 3IVF cycle results in study and control groups.

Study group

(n = 28)

Control group

(n = 61)

P

GnRH protocol type <0.05a

Long agonist 2(7.1%) 27(44.3%)

Short agonist 13(46.4%) 20(32.8%)

J.S. Younis et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 166 (2013) 65–69 67

COH and IVF-ET results are summarized in Table 3. It is clearlyshown that women in the study group required higher dosage andlonger duration of gonadotrophins as compared to controls. Asexpected, the mean numbers of follicles, oocytes, and embryoswere significantly lower in the study as compared to the controlgroup. A similar number of embryos were transferred to the uterusin the fresh cycles in the two groups. Clinical pregnancy rate percycle was higher in the control group as compared to the studygroup with borderline significance (P = 0.06), corresponding to19.7% versus 7.1%, respectively

Table 4 summarizes the thrombophilic work-up undergone byboth groups during the study. The prevalence of protein C, proteinS, anti-thrombin III, acquired activated protein C resistance (APCR),anti-cardiolipin antibodies (ACL, Ig G and Ig M), lupus antic-oagulants (LAC), factor VIII, and factor V Leiden and ProthrombinG20210A (PT) mutation was similar between the two groups.Moreover, the prevalence of at least one thrombophilia and two ormore thrombophilias present were similar between the study andcontrol groups. The prevalence of methylenetetrahydrofolatereductase (MTHFR) mutation was also similar between the groupsbut it was not analyzed as a thrombophilic risk factor.

In addition, employing logistic regression analysis models, takinginto account chronological age, no correlation was found betweennumber of oocytes retrieved and thrombophilia presence (P > 0.05).Likewise, Pearson correlation tests did not show a significantassociation between number of oocytes retrieved and presence of aspecific thrombophilia or any thrombophilia tested (P > 0.05). Theonly correlation with number of oocytes retrieved was found with PTmutation, but only 3 cases with PT mutation were found in all

Table 2Early follicular ovarian reserve tests in the study and control groups.

Study group

(n = 28)

Control group

(n = 61)

Pa

FSH (mIU/mL) 9.3 � 4.8 7.1 � 2.0 <0.05

LH (mIU/mL) 4.9 � 3.3 4.8 � 3.1 0.87

FSH/LH ratio 2.2 � 1.6 1.9 � 1.1 0.26

E2 (pg/mL) 59.4 � 32.7 48.9 � 18.3 0.12

Progesterone (ng/mL) 1.3 � 2.6 0.5 � 0.3 0.15

Antral follicle count 4.3 � 3.5 9.3 � 5.1 <0.001

Total ovarian volume (cm3) 9.6 � 4.5 17.1 � 6.4 <0.001

Mean ovarian volume (cm3) 5.0 � 2.1 9.0 � 3.1 <0.001

a Two-independent sample t-test.

women included in the study. Furthermore, basal ovarian reservetests, including early follicular serum FSH level, AFC and ovarianvolume, did not differ between women with at least onethrombophilia and those with no thrombophilia.

To examine the impact of thrombophilia on premature poorovarian response, two age cut-off points were elected. The twosub-groups of women �37 and �35 years of age were separatelyanalyzed. Among the 89 women included in the study, 61 womenwere 37 years of age or less. Eleven of these women retrieved �3oocytes while 50 retrieved �4 oocytes. The incidence of a single aswell as �2 thrombophilias was similar between the groups. Amongthe 89 women included in this study, 48 were 35 years of age orless. Five of these women retrieved �3 oocytes while 43 retrieved�4 oocytes. All five women had a previous cycle of poor ovarianresponse and an abnormal ovarian reserve test in accordance withnew ESHRE criteria of poor response [28]. Interestingly, theincidence of at least one thrombophilia in this sub-group wassignificantly higher in the poor ovarian response group ascompared to the controls, corresponding to 80% and 30.4%,respectively (Z = 2.20, P = 0.014). Furthermore, the incidence of�2 combined thrombophilia was significantly higher in the study

Antagonist 13(46.4%) 14(22.9%)

Gonadotropin duration (days) 12.3 � 4.0 10.7 � 1.9 0.0542b

Gonadotropin dosage (IU) 4858 � 2156 2808 � 846 <0.001b

Number of follicles � 14 mm 3.9 � 3.0 10.6 � 5.1 <0.001b

Number of oocytes

Total 1.6 � 1.2 10.8 � 4.9 <0.001b

MII oocytes 1.5 � 1.0 8.6 � 4.1 <0.001b

Number of 2PN zygotes 1.3 � 1.1 5.4 � 3.0 <0.001b

Number of embryos

Total 1.3 � 1.0 5.7 � 3.4 <0.001b

Transferred 0.8 � 0.9 2.0 � 1.0 <0.001b

Frozen 0.1 � 0.4 3.6 � 3.4 <0.001b

Clinical pregnancy/cycle 2 (7.1%) 12 (19.7%) 0.063c

a Chi square test for crosstab data.b Two-independent sample t-test.c Z-test for two proportions.

Table 4Thrombophilia results in the study and control groups.

Study group (n = 28) Control group (n = 61) Pa

Free protein S antigen �60 u/dl 0 0 NS

Protein C activity �70 u/dl 1(3.6%) 0 NS

Anti-thrombin III �70 1(3.6%) 0 NS

Activated protein C resistance – acquired �2.15 5(17.9%) 5(8.2%) NS

Anti-cardiolipin Abs Positive 5(17.9%) 6(9.8%) NS

Lupus anticoagulants Positive 1(3.6%) 0 NS

Factor VIII �180 u/dl 4(14.3%) 6(9.8%) NS

Factor V Leiden Heterozygote 3(10.7%) 10(16.4) NS

Homozygote 0 1(1.6%)

Prothrombin G20210A mutation Heterozygote 1(3.6%) 2(3.3%) NS

Homozygote 0 0 NS

At least one thrombophilia 11(39.3%) 20(32.8%) NS

�2 Thrombophilias 6(21.4%) 14(23.0%) NS

MTHFR mutationb Heterozygote 8(28.6%) 24(39.3%) NS

Homozygote 3(10.7%) 13(21.3%) NS

a Z-test for two proportions.b MTHFR mutations were not considered as thrombophilia.

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as compared to the control group, corresponding to 60% and 25.6%,respectively (Z = 1.60, P = 0.05). The thrombophilias encounteredin these five women were as follows: acquired APCR, high FVIIIlevel, ACL antibodies and LAC in case number 1; FVL (heterozy-gote), PT mutation (heterozygote) and ACL antibodies in casenumber 2; acquired APCR and high FVIII level in case number 3;ACL antibodies in case number 4; no thrombophilia was detectedin case number 5.

4. Comments

Our prospective study, performed in an ART setting, impliesthat thrombophilic risk factors have no association with poorovarian response in the general infertile population. The incidenceof all thrombophilic risk factors evaluated was similar betweenwomen retrieving �3 oocytes and those retrieving �4 oocytes.Furthermore, the incidence of at least one single or combined (�2)thrombophilias was also similar between the two groups. Inaddition, logistic regression analysis did not show a correlationbetween number of oocytes retrieved and thrombophilia presence.Likewise, Pearson correlation tests did not show a significantassociation between number of oocytes retrieved and the presenceof a specific thrombophilia or any thrombophilia tested.

Additionally, basal ovarian reserve tests results did not differsignificantly between women with thrombophilia and those withno thrombophilia, adding to our conclusion that poor ovarianresponse is not correlated with thrombophilic risk factors.

Our results are in accordance with those published recently byRicci et al. in an ART setting [29]. The mean number of oocytesretrieved was shown to be similar between those with and otherswithout a thrombophilic risk factor. However, the main objectiveof Ricci et al’s study was to compare IVF-ET outcome and risk ofcomplications between thrombophilic women and controls.Moreover, it included only factor V Leiden and PT mutations forevaluation. To the best of our knowledge our study is the first thathas targeted the association between ovarian aging and thrombo-philia in an ART setting (PubMed.com search engine January 1993–June 2012). Furthermore, our study evaluated a broad range ofthrombophilic risk factors.

Earlier studies suggested an association between thrombo-philic risk factors and ovarian aging. Early onset of naturalmenopause has been reported to be associated with vascular-related candidate genes such as factor V Leiden [19,20], highlevels of factor VII [21,22] and apolipoprotein E gene mutation[19,30,31]. Other investigated vascular-related genes, like nitricoxide synthase, clotting factor II and angiotensinogen, were not

found to be associated with the development of early meno-pause [19,32–34].

In a recent large-scale candidate gene association study of ageat natural menopause, common genetic polymorphisms ofbiologically plausible pathways and related phenotypes wereinvestigated [35]. Among these candidate genes, 32 genes wererelated to thrombophilia and vascular homeostasis pathways.None of these genes was found to be significantly associated withage at natural menopause, negating an association betweenthrombophilia and ovarian aging, and supporting our findings inthis study.

Nevertheless, when evaluating the methodology of this studyin depth it is important to note that women reporting age atmenopause younger than 40 or older than 60 years of age wereexcluded. Moreover, age at natural menopause was self-reportedand ascertained only by questioners [35]. These factors mayinfluence the interpretation of results, and cause bias whenarriving at final conclusions. Alternatively, conducting a prospec-tive study during reproductive age, in an ART setting andemploying ovarian reserve tests, COH and IVF-ET results as isthe case in our study, seems to be more appropriate to study thelink between ovarian aging and the vascular homeostaticpathway.

Our finding that women with premature low ovarian reserve,manifested by poor ovarian response, might have an associationwith thrombophilic risk factors is interesting and deserves furtherinvestigation. However, the small sample size of five women in thestudy group of young women �35 years of age precludes a definiteconclusion concerning the association between premature ovarianaging and thrombophilia.

We have to emphasize that our study was planned as aprospective cohort study with a total of 89 infertile unselectedwomen admitted consecutively for ART treatment. The incidenceof premature ovarian aging is estimated to be about 10% in womenof reproductive age [6–8], very close to our finding of 5/48 (10.4%),in this sub-group. A prospective targeted study in women withpremature low ovarian reserve is needed to substantiate thisfinding and evaluate the association with thrombophilia.

If this finding is substantiated, it might be suggested thatvascular compromise might contribute to the development ofpremature ovarian aging rather be the result of it and thatthrombophilia is implicated in this process. The free radical andoxidative stress theory is one of the most popular explanations forhow aging occurs at the biochemical and molecular levels [36]. Inthis regard it might be proposed that homeostatic compromise ofthe ovarian vasculature may lead to accumulation of oxidant

J.S. Younis et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 166 (2013) 65–69 69

stimuli that would increase vulnerability to oxidative stress andcontribute to ovarian senescence.

In conclusion, in a prospective preliminary cohort study of 89infertile women undergoing IVF-ET treatment, thrombophilicrisk factors were not associated with poor ovarian response,defined as �3 oocytes on retrieval day. Whether premature lowovarian reserve in women below 35 years of age is linked tothrombophilia and vascular homeostasis pathway remains to beestablished.

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