Luteal progesterone level correlated with immunotherapy success of patients with repeated implantation failures

Immunotherapy using PBMC administration demonstrated relatively its effectiveness to treat RIF patients but it still unclear to explain some miscarriages. Luteal progesterone level (LPL) issued from corpus luteum after embryo implantation stage could be informative basis data to personalize immunotherapy for RIF patients predicting clinical outcomes. This randomized controlled study included 70 patients undergoing ICSI program presenting at least 3 RIF: 39 for Control of untreated patients and 31 for PBMC-test concerning treated patients with immunotherapy. For PBMC-test group, Peripheral Blood Mononuclear Cells (PBMCs) were isolated from patients on ovulation induction day and cultured three days to be administered to intrauterine cavity of patients two days before fresh embryo transfer. LPL was analyzed at day 15 after embryo transfer and clinical outcomes were calculated including implantation, clinical pregnancy and miscarriage rates. Clinical outcomes were doubly improved after immunotherapy including implantation and clinical pregnancy rates comparing Control versus PBMC-test (10% and 21% vs 24% and 45%). In the other hand, this strategy showed an increase over double in LPL (4ng/ml for Control vs 9ng/ml for PBMC-test) while the latter was correlated to clinical pregnancy. Bypassing the effectiveness of this immunotherapy approach for RIF patients, it is directly correlated to LPL proving the interactive reaction between immune profile of the treated patients and progesterone synthesis by corpus luteum.

Though in vitro fertilization (IVF) success is generally limited to 30% depending on in order to assure embryo invasion in the maternal endometrium without rejecting the 6 fetal allograft. Furthermore, it is already known that progesterone (P4) is one of the 7 most important implantation/pregnancy success keys for its effects on the endometrium 8 and early pregnancy survival while its removal results in miscarriage [1][2][3]. 9 P4 is a hormonal key to modulate the maternal immune system by reducing natural 10 killer (NK)-cell activity [4], inhibiting cytotoxic T-cell activity [5], increasing HLA-G 11 production in trophoblast cells [6], increasing suppressor-cell levels [7] and as special 12 mechanism, it is able to induce lymphocyte-blocking proteins production such as 13 progesterone-induced blocking factor (PIBF) [8][9][10][11]. Generally, its anti-inflammatory 14 effect reported by several studies showing that it is essential to modify the cytokine 15 response from pro-inflammatory profile presented by Th1 during embryo implantation 16 to anti-inflammatory profile presented by Th2 for pregnancy maintain [12-16, 18, 19]. 17 Thus, Th1/Th2 unbalance could explain implantation failures in some patients with 18 RIF, RPL or recurrent miscarriages (RM) [20][21][22]. 19 Indeed, with special interest on immunology of reproduction, Yoshioka et al. [23] 20 was the first team which could to realize immunotherapy for patients with repeated IVF 21 failures based on intrauterine administration of peripheral blood mononuclear cells 22 (PBMC). Then, several studies developed this novel approach with some modifications 23 on PBMC preparation protocol, including fresh or frozen cycles, embryo day 3 or 24 blastocyst transfer, patients with at least 2, 3 or 4 RIF in order to improve significantly 25 clinical outcomes and decrease the miscarriage rates [22,24,25]. 26 Furthermore, PBMC immunotherapy could to be efficient for some RPL cases and 27 avoiding miscarriages [22,26,27] despite lack of clear definition toward RIF and 28 RPL [28]. This issue led us to wonder about mechanism of PBMC immunotherapy by 29 what it could modulate maternal immune response homing Th1/Th2 balance required 30 for implantation and pregnancy stage in order to manage RIF and RPL clinical cases 31 and its effect on luteal P4 synthesis. Indeed, it is known that P4 is an indispensable 32 factor for endometrium decidualization and for early stage of clinical pregnancy to 33 prepare an adequate immune environment for fetus and low LPL results abnormal 34 ongoing pregnancy [18,19,[29][30][31][32]. 35 Forward, it is increasingly clear that embryo implantation is dependent in one side 36 on immune local mechanisms and in another side on endocrine mechanisms related to 37 luteal P4 synthesis while their interactive reaction is still kept into question for 38 pregnancy achievement. For this reason, while our previous work [22] was based on the 39 implementation of PBMC immunotherapy for RIF patients and proving its efficiency 40 tripling the clinical outcomes, the present work was more focused on demonstrating the 41 correlation between LPL and immunotherapy success highlighting the interactive 42 reaction between luteal P4 synthesis by CL and immune system. Patient's selection and study design 53 This was a prospective randomized study over two years conducted in Anfa Fertility 54 Center including 70 couples who attended IVF program with at least 3 RIF without 55 female age limit while 48 patients of them were less than 40 years old. In the selected 56 couples, women had unremarkable clinical history and comparable clinical features and 57 embryological outcomes (Table 1). All women received the same antagonist ovarian 58 stimulation protocol [22] to minimize the effect of other parameters. Indeed, the whole 59 lot was divided into two groups; treated group with PBMC immunotherapy 60 (PBMC-test, n=31) and the control group without treatment (Control, n=39). Results are expressed as n, n(%) or mean ± standard deviation (SD). A statistic significant difference is considered when P<0.05 (n). P≥0.05 is not significant (ns). AMH, estradiol and progesterone were measured on day 2 of the cycle and the endometrial thickness was evaluated in day of oocyte retrieval. Cleavage rate was calculated relatively to embryos at day 3 by 2 pronucleus.

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Embryos produced by ICSI [22] were cultured up to day 3. Adequate embryo quality 63 (good quality embryos; A+B) was defined based on the presence of uniformly sized and 64 shaped blastomeres and fragmentation lower or equal to 10%. One or two good quality 65 embryos were transferred in utero using a Frydman catheter (CCD Laboratories, Paris, 66 France). The implantation success (observation of the embryo sac) was assessed by 67 ultrasound imaging and calculated relative to the number of transferred embryos. After antagonist ovarian stimulation protocol, a blood sample is scheduled on the day of 74 ovulation induction to isolate PBMCs using a separation protocol based on Ficoll.

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PBMCs are well prepared after a culture for 72 h and then transferred to the patient in 76 utero two days before embryo transfer as it was elucidated by Madkour et al. [22]. After 77 embryo transfer, patients receive oral Utrogestan (200 mg×2/day) for luteal support.
In the course of our study, the included patients underwent the LPL analysis at day 79 15 after embryo transfer to reflect the P4 synthesis by CL after implantation using the 80 serum for the first pregnancy test for β-hCG assay. Indeed, the LPL analysis was  --Results are expressed as n, n(%) or mean ± standard deviation (SD). A statistic significant difference is considered when P<0.05 (n). P≥0.05 is not significant (ns). Power 1-β (β is error type II) is calculated basing on difference of mean values between two groups (PBMC-test vs Control) with α= 0.05 (α is error type I). Power value is considered highly important when it is above 80%. r-correlation was calculated relatively to LPL depending on clinical pregnancy rate in each group of PBMC-test and Control, and it is considered significant when P<0.05 and non significant when p≥0.05.The implantation rate is expressed as the ratio between the number of embryonic sacs and the total number of transferred embryos; the miscarriage rate is expressed relative to the number of clinical pregnancies. LPL (Luteal progesterone level) was measured at day 15 after embryo transfer.

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Despite there is not clear differential clinical diagnostic to differ between RIF and RPL, 100 over 75% of pregnancy failures are due to implantation failures [33]. Whatever 101 controversies regarding RIF and RPL clinical definition, hypothetically in our previous 102 study [22] it was suggested that RIF is due to pro-inflammatory (Th1) deficiency while 103 RPL is due to Th1 persistence inhibiting the anti-inflammatory (Th2) release. 104 Therefore, in this current study following the results of our previous work [22] PBMC 105 immunotherapy was efficient for RIF patients since their second implantation failure to 106 double their chance to conceive. Nevertheless, we are not the only team who are 107 prescribing this kind of treatment to patients with RIF or generally with IVF failures. 108 Yoshioka et al. were the pioneers in this approach application while the research teams' 109 followers could to involve some technical modifications in PBMC preparation 110 protocol [23]. Some could to prove the efficiency of hCG supplementation on PBMC 111 culture for 72h [22,24] or trying to minimize latter to 24h [26,27,34] while others were 112 more focused on the efficiency of CRH [25]. All these technical adaptations were 113 occurred in order to enhance at maximum the function of PBMC and their cytokines 114 secretions to activate thereafter the maternal immune system into endometrium after an 115 intrauterine administration and be ready for embryo implantation. Indeed, as expected, 116 implantation rate after PBMC immunotherapy was over double compared to control 117 (24% vs 10%; table 2) and the result was similar to other studies with interval 21-25% 118 for treated patients [22-24, 26, 27]. 119 Nevertheless, it was commonly accepted that with insufficient P4 production causing 120 miscarriages could be solved simply by an exogenous P4 administration in order to 121 regulate the inflammatory mediators of pregnancy and even for patients undergoing IVF 122 process in fresh or frozen cycles to improve clinical outcomes prior embryo 123 transfer [29,35,36]. Indeed, P4 presents an anti-inflammatory action enhancing Th2 124 cytokines production to maintain pregnancy [37,38].  (Table 2). However, 4 ng/ml of LPL was not correlated to clinical pregnancy 130 rates for RIF patients in control group (r=0.16). This observation allowed us to 131 conclude that certainly even with P4 importance to maintain clinical pregnancy, this 132 latter could be occurred. Moreover, it explained why P4 supplementation treatments 133 kept into question their efficiency for RIF, RPL and RM patients despite the maternal 134 immune system is dysregulated led toward Th1 or Th2 [21,29,39,40]. In the opposite 135 side, when this latter is probably trying to turn back its balance via PBMC 136 immunotherapy which acted not only into endometrium but also in CL to produce more 137 the P4, LPL became correlated to clinical pregnancy. This hypothesized interactive 138 reaction between LPL issued from CL and PBMC administrated into endometrium, is 139 realized in order to assure the embryo implantation and pregnancy maintain homing 140 Th1/Th2 balance (Fig 1). 141 Furthermore, luteal P4 would regulate the uterine level synthesis of CSF-1, cytokine 142 essential for the vascularization of the endometrium and to maintain pregnancy by 143 increasing just before implantation to achieve a peak tripled to day 15 of 144 pregnancy [41,42]. Our results show a 63% increase in the synthesis of the luteal 145 progesterone in pregnant patients treated with PBMC which joins perfectly the 146 observed effect of PBMC on clinical pregnancy rate in our study. It seems that this 147 effect is mediated by Th2 cells secreting IL4 and IL10 able to optimize the recruitment 148 of leukocytes for VEGF secretion in CL [13]. The latter is better vascularized release P4 149 production [43].

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Te PBMC effect could not be certainly over early pregnancy stage to balance 151 maternal immune system and LPL while P4 will be placental and the ongoing 152 pregnancy until delivery will be more influenced by fetus and genetic reproductive 153 function. May be for this reason, PBMC immunotherapy is less effective to avoid 154 miscarriages as showed in our study with non-significant difference in miscarriage rate 155 (21% for PBMC-test and 75% for control; Table 2) and confirmed by others [22,26]. 156 The paramount function of PBMC is to provide trophic support for endometrium to 157 Fig 1. Hypothesis of interactive effect of PBMC immunotherapy and luteal progesterone level for implantation and pregnancy success. After intrauterine administration PBMC, the Th1 / Th2 balance towards Th1 tends to ensure ignition shift by secreting pro-inflammatory cytokines and several growth factors primarily VEGF inducing vascularization in one hand into endometrium to prepare for embryo invasion embryo and in the other hand into CL in order to increase luteal progesterone level (LPL). All these immune-endocrine factors are limited in closest communication circle with mutual interactive modulation to ensure the embryo implantation. Thereafter, an increased LPL can induce immunomodulation by promoting T cells differentiation into Treg and secreting PIBF as an immunosuppressor factor that promotes the Th1/Th2 balance to Th2 anti-inflammatory system ensuring immunotolerance of allograft "embryo". Thus, Th2 cytokines secretion involved in CL maturation can eventually to increase more LPL required for pregnancy maintain. be decidualized and for formation of dense vascular network in CL to produce more P4 158 that is essential for pregnancy maintain. However, perturbations of immune-endothelial 159 cell crosstalk within the ovary during the peri-conceptional period are likely to be 160 pivotal in luteal insufficiency in women. This issue could provide more therapeutic 161 trends to enhance luteal function through the targeting of immune system.