Mohammed, Amal Abdulwahid
(2019)
The mechanisms underlying uterine receptivity in pigs and women.
PhD thesis, University of Nottingham.
Abstract
Successful implantation and pregnancy require synchronisation between a fully receptive endometrium and a well-developed embryo. In assisted reproductive technologies (ART), the embryo is created in artificial conditions while endometrial receptivity is affected by ovarian stimulation treatment. Therefore, one or both of these factors may be major contributors to embryo loss. Many supplementation regimes have been utilised to optimise this synchronisation and to provide luteal phase support in ART cycles.
In this study, the use of progestogen and oestradiol supplementations in women undergoing ART was assessed using a systematic review and meta-analysis approach. In addition, porcine models were used to evaluate the communication between embryo and endometrium during the peri-implantation period.
Meta-analysis studies were performed for women undergo ART cycles with fresh or frozen embryo transfer and in intrauterine insemination (IUI). All available trials that compared the effects of progestogen treatments alone or with oestradiol were included and all the studies did not meet the eligibility characteristics were excluded. In fresh embryo transfer, 211 studies with 29,357 women undergoing IVF/ICSI cycles were analysed. The use of progestogen supplementation improved the clinical pregnancy rate versus no supplementation (p<0.001 from 18% to 35% in treated cycles).
Starting progestogen supplementation 1-1.5 days after oocyte retrieval was more beneficial (p<0.001, 41%) than any other start time. Using the intramuscular route was significantly more beneficial (p<0.001, 43.8%) to clinical pregnancy rates than the vaginal route (35.8%). This meta-analysis study also showed that adding oestrogen to progestogen was beneficial (p<0.01) for clinical pregnancy rate. In frozen embryo transfer, 30 studies (5146 women) were used to assess clinical pregnancy rate after progestogen supplementation. Regardless of the method used for endometrium preparation, progestogen supplementation as luteal phase support was beneficial (p<0.01) for clinical pregnancy outcome. In intrauterine insemination (IUI) cycles, there were 14 studies (1555 women) included in the analysis, which illustrated that using progestogens increased (p<0.01) clinical pregnancy rate after using gonadotrophins for ovarian simulation.
The first porcine study investigated the relationship between embryo development and number, luteal function and oestradiol concentration in uterine flushes on days 3-5, 11-12 and 13-14 of pregnancy (47 pigs in total). The number and weight of corpora lutea were similar in the animals that had a low (≤9) or high (>9) number of embryos. The total luteal progesterone content on pregnancy day 3-5 was significantly higher (p<0.05) in animals with a high number of embryos compared with those with a low number of embryos, while there were no differences on pregnancy days 11-12 and 13-14. Moreover, the result demonstrated that on pregnancy day 11-12, higher oestradiol levels in uterine flushes were significantly associated (p<0.05) with the presence of embryos at the filamentous stage. However, there was no association between the number of embryos and the uterine flush level of oestradiol on days 3-5 and 11-12 of pregnancy.
Immunohistochemistry demonstrated that the preimplantation disappearance of progesterone receptors (PGR) in luminal epithelial cells was associated with an increased expression of integrin αvβ1 (ITGA5/B1) in pregnant sows on days 11-12 and 13-14 of pregnancy. In addition, stanniocalcin 1 (STC1) protein was localised to the luminal epithelial and stroma cells of the endometrium.
On day 11-12 of pregnancy (n=6), there was no significant association between the endometrial progesterone content (high versus low) and STC1, osteopontin (OPN) or mucin 1 (MUC1) expression in the endometrium. Explant culture was used to study the effects of progesterone and oestradiol treatment on STC1, OPN and MUC1 expression in endometrium from pregnant sows (n=8) on days 3-5, 11-12 and 13-14. To assess the regulation of STC1, OPN and MUC1 by steroid hormones, an endometrial explant tissue culture system was developed in which tissue structure, cell viability and PGR expression were maintained over several days without inducing high levels of apoptotic cell death.
Endometrial explant tissues were treated with 1) progesterone (0, 10 and 100 ng/ml) or 2) oestradiol (0, 0.1 and 1 ng/ml) in the absence or presence of 10 ng/ml progesterone in a 3 × 2 factorial design. Tissue was collected after 0, 48 and 96h in culture to determine the protein expression of STC1, OPN and MUC1 by Western blot. There was no evidence to support the hypothesis that STC1 expression was influenced by progesterone or oestradiol treatment. OPN protein was expressed at all experimental times, while MUC1 was only expressed at the start of culture.
A novel culture system was established to investigate angiogenesis in the porcine endometrium (non-pregnant sows in mid-luteal phase, n=4). This was utilised to examine the effect of STC1 (0, 5 and 50 ng/ml), prostaglandin F2α (PGF2α; 0, 10 and 100 ng/ml), prostaglandin E2 (PGE2: 0, 10 and 100 ng/ml) and uterine flushes from pregnant sows at day 11-12 and 13-14 (0, 20 and 200 µg/ml total protein) on endometrial endothelial cell (EC) network formation. These were investigated in basal conditions or with the addition of pro-angiogenic factors (VEGFA plus FGF2). STC1 increased the formation of EC networks significantly (p<0.001) and PGE2 had a positive effect on EC network formation in basal conditions (p<0.05). While PGF2α did not show any significant impact. In contrast to expectations, uterine flushes from both pregnancy days had a negative effect on EC formation with 200µg/ml total protein blocking all EC formation in basal and angiogenic factors conditions.
In summary, progesterone supplementation has a fundamental positive impact on pregnancy outcome in women undergoing ART. More importantly, revised optimal time of starting supplementation (the day after oocyte retrieval) and route of progesterone (intramuscular) were identified. The communication between the endometrium and conceptus tissue is fundamental for successful pregnancy. The appropriate timing of uterine receptivity is dependent on the synchronised secretions from the ovary and embryo. These secretions are likely to regulate endometrial angiogenesis and thereby influence the likelihood of attachment and implantation occurring. Indeed, this study demonstrated that STC1 promoted angiogenesis to a greater degree than VEGFA/FGF2 and might be a therapeutic strategy by which endometrial receptivity could be improved.
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