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Original Studies |
Instituto Valenciano de Infertilidad (C.S., A.M., J.G.-V., C.M., A.P.) and the Department of Pediatrics, Obstetrics, and Gynecology (C.S., J.R., A.P.), Valencia University School of Medicine, 46020 Valencia, Spain; and Hammersmith Hospital (G.N.), London, United Kingdom
Address all correspondence and requests for reprints to: Dr. Carlos Simón, Instituto Valenciano de Infertilidad, Guardia Civil 23, 46020 Valencia, Spain. E-mail: csimon{at}interbook.net
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The present trend in humans is to transfer embryos at the blastocyst stage, as in laboratory and domestic animals, because this leads to high implantation and pregnancy rates (3, 4, 5) and also because it is a more physiological approach (the human embryos enter the endometrial cavity only after day 5 at the morula-blastocyst stage). The main problem with blastocyst transfer in humans is to develop consistent, safe, and effective culture systems to obtain an adequate percentage of blastocysts.
The concept of improved human preimplantation development and implantation ability by coculturing embryos in the presence of another cell type (feeder cells) has led to the development of the coculture system. Multiple cell types have been used for this purpose, ranging from human reproductive tissues, such as oviducts (6, 7), endometrium (8, 9), oviduct-endometrial sequential coculture (10), and cumulus-granulosa cells (11, 12, 13, 14), to nonhuman cells (15) or nonhuman cell lines (16, 17, 18), and even cells from ovarian carcinoma (19). As a consequence, the embryonic effects reported using this technology are cell, tissue, and species nonspecific. The suggested beneficial effects of cocultures include the secretion of embryotrophic factors such as nutrients and substrates, growth factors, and cytokines (for review, see Ref. 20) and the removal of potentially harmful substances such as heavy metals, ammonium, and free radical formation, detoxifying the culture medium. The main objective is to increase the metabolic chances of the human embryo to achieve the blastocyst stage and implant.
Unfortunately, there is no general agreement on the efficacy of different coculture systems (20); not even has the utility of the coculture itself compared to that of a chemically defined medium (20, 21) been proven. Even with the most extended coculture system, i.e. Vero cells, results in randomized studies are discrepant (22, 23). Further, when coculture systems are employed, in addition to the aim of high yield production of viable blastocysts, there are other important end points, such as medical, ethical, and practical feasibility.
Data from a number of studies provide convincing evidence of a chemical dialogue between the developing embryo and the maternal endometrium (24, 25). This embryonic-endometrial cross-talk may be beneficial not only to improve the blastocyst rate, but most importantly for the activation of specific paracrine molecules in a timely manner that may improve the chances of implantation of the embryo (26). Recently, our group has demonstrated that a coculture system with human endometrial epithelial cells (EEC) is beneficial to the human blastocyst because of the induction of secretion of embryonic paracrine molecules (27). Moreover, the human embryo cocultured under these conditions improves uterine receptivity by increasing EEC adhesion molecules such as the ß3 integrin subunit (28). The objective of this work was to develop clinically this basic concept so as to improve the chances of implantation and pregnancy of patients with implantation failure (IF) undergoing either IVF (with their own oocytes) or oocyte donation (with donated oocytes) compared to a routine day 2 transfer.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Based on our previous works (27, 28), we have developed a clinical program in which embryos were cocultured with autologous EEC (AEEC) until the blastocyst stage and then transferred back to the patient. To assess the safety of the embryo coculture with AEEC, we first tested whether primary cultures of EEC obtained from patients with IF were detrimental to embryonic development compared to endometrium obtained from fertile patients by morphological (scanning electron microscopy) and biological tests. Second, to make this model feasible and convenient, an endometrial freezing and thawing method was developed and tested to perform primary EEC cultures from the patient’s endometrium obtained in the luteal phase of a previous cycle.
To investigate the clinical potential of this technique in improving implantation, coculture with AEEC was clinically applied to patients with IF, defined as at least three previously failed cycles with three or four good quality embryos transferred in either IVF (with their own oocytes) or oocyte donation (with donated oocytes), and compared to a routine day 2 transfer in patients with IF.
Patients and institutional approval
This project was approved by the Instituto Valenciano de Infertilidad review board on the use of human subjects in research. The experimental design took the form of volunteer assignment as opposed to randomization, because the latter was disqualified by the review board due to previous experimental findings suggesting that coculture of embryos with EEC would improve embryo viability. However, all patients were provided with background information about the treatment groups.
This study includes a total of 168 IVF cycles and 80 oocyte donation
cycles undergoing coculture with AEEC and blastocyst transfer for IF in
our unit during the period January 1, 1996 through March 31, 1998. The
inclusion criteria were patients undergoing IVF or ovum donation with
at least 3 previous cycles failed with 3–4 good quality embryos
transferred. Controls were composed of 20 IVF cycles and 15 ovum
donation cycles with IF, in which a day 2 embryo transfer was performed
during the same period of time. The infertility etiology for undergoing
IVF and ovum donation were similar in both controls and AEEC coculture
groups (Table 1
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Endometrial biopsies were obtained from patients undergoing IVF (in the luteal phase of the previous spontaneous cycle) and fertile patients on day LH 6 after LH titration and ultrasound scanning to demonstrate corpus luteum formation. In hormone replacement therapy mock cycles, biopsies were taken 8 days after progesterone (P) administration. Endometrial tissue were rinsed with DMEM (Sigma Chemical Co., Madrid, Spain) and placed into 1 mL freezing medium composed of DMEM (700 µL) plus 200 µL charcoal-stripped inactivated FBS (HyClone Laboratories, Inc., Logan, UT) plus 100 µL dimethylsulfoxide. The freezing procedure was performed for 2 h at -80 C, and the cryovial was subsequently stored in liquid N2. To thaw, the cryovial was incubated at 37 C for 3 min, and the endometrial culture was initiated immediately.
Endometrial culture
Endometrial samples were minced into small pieces of less than 1 mm and subjected to mild collagenase digestion. Endometrial stromal cells and EEC were isolated as previously described (29, 30). Epithelial cells were cultured and grown to confluence in steroid-depleted medium; 75% DMEM and 25% MCDB-105 (Sigma Chemical Co.) containing antibiotics, supplemented with 10% charcoal-dextran-treated FBS and 5 µg/mL insulin (Sigma Chemical Co.). The homogeneity of cultures was determined by morphological characteristics and was verified by immunocytochemical localization of cytokeratin, vimentin, and CD68 antigen as previously described (30). Confluence was reached in 3–5 days, then growth medium was replaced by IVF/S2 (1/1) (Scandinavian IVF, Göteborg, Sweden), and single human embryos were cocultured in the EEC monolayer.
Fixation and scanning electron microscopy
For fixation, 1 mL 1% gluteraldehyde (Sigma Chemical Co.) in PBS was added to the EEC monolayers obtained from fresh and frozen endometria in the presence or absence of blastocyst-conditioned medium. Samples were stored in the fixative at 4 C for several days until they were processed. For scanning electron microscopy, the specimens were dehydrated in alcohol series and then dried according to the critical point method using CO2. After drying, EEC monolayers were mounted on the specimen holder, sputter-coated with gold (14 nm thickness), and observed under accelerated voltage of 10.0 kV at a short working distance in a Cambridge Stereoscan 360 scanning electron microscope (Cambridge Instruments, Cambridge, MA). For measurements, the screen magnification was increased to 20,000, and three representative areas of 4 µm2 were examined for each specimen. All specimens were processed together.
Mouse embryo assay
Two-cell embryos were flushed from the oviducts on day 2 of pregnancy from 8-week-old Swiss CFLP mice (Harlan Interfauna Iberica, Barcelona, Spain) as previously described (31). Embryos were cultured in the presence of IVF, M3, or Hatch-50 alone or cocultured with the same medium in the presence of EEC obtained from either fertile patients or patients with implantation failure. Results were expressed as the percentage of two-cell mouse embryos that reached the blastocyst stage after 72 h in culture.
Clinical IVF protocol
The ovarian stimulation protocol using GnRH analogs and gonadotropins has been previously described (32). Briefly, a long protocol was used for pituitary desensitization with administration of leuprolide acetate (1 mg/day, sc; Procrin, Abbot S.A., Madrid, Spain), starting in the luteal phase of the previous cycle. Serum estradiol (E2) levels below 60 pg/mL (conversion factor to Systeme International unit, 3.671) and negative vaginal sonographic scan were used to define ovarian quiescence. Human menopausal gonadotropins (Pergonal, Serono Laboratories, Inc., Madrid, Spain; Fertinorm, Serono Laboratories, Inc., Madrid, Spain) were administered for ovarian stimulation, and routine criteria for hCG administration (10,000 IU; Profasi, Serono Laboratories, Inc., Madrid, Spain) were used. Oocyte retrieval was performed 36–38 h after hCG administration. The standard IVF procedure has been previously described (32). A good quality embryo is an embryo with even blastomeres and no fragmentation (grade I) or with uneven blastomers and less than 20% fragmentation (grade II).
Donor characteristics and ovum donation protocol for recipients
Oocytes were obtained from a total of 69 patients. Forty-four infertile patients undergoing IVF (polycystic ovary syndrome, n = 17; idiopathic infertility, n = 10; male infertility, n = 9; tubal infertility, n = 8) and 25 fertile women. The mean age was 31.6 yr.
In recipients with ovarian function, GnRH analogs (leuprolide acetate, 1 mg/day, sc) were administered in the secretory phase of the previous cycle. Hormonal replacement started on day 1 of the cycle with administration of estradiol valerate (EV; Progynova, Schering AG, Madrid, Spain; 2 mg/day on days 1–8; 4 mg/day from days 9–11; and 6 mg/day from day 12 on). After 13 days of EV administration, recipients were ready to receive the donation, and they waited until a donation became available (33). On the day of oocyte recovery, 800 mg/day natural micronized P were administered vaginally to the recipient when embryo transfer was performed at 48 h (day 2 transfer). For blastocyst transfer, P in the recipient was started 24 h after oocyte retrieval in the donor. The regimen of 6 mg/day EV and 800 mg/day P was maintained for 15 days, after which urinary hCGß analysis was performed. In the case of a positive result, EV was increased to 8 mg/day, and P was maintained at the same dosage until day 80 of pregnancy.
Human embryo coculture with AEEC and blastocyst transfer
Forty-eight hours after insemination, two to four-cell embryos were cocultured individually on the AEEC monolayer. At this time, embryos were grown in 1 mL IVF/S2 (1/1) until they reached the eight-cell stage, and then cultured with S2 until the blastocyst stage. Embryonic development was checked daily, and conditioned media were changed every 24 h. During the last 24 h of culture, blastocyst development was recorded using a video time-lapse system (Life Science Resources Ltd., Cambridge, UK). On day 6, blastocysts were transferred using a Frydman catheter.
Statistical analysis
Data were expressed as the mean ± SEM. For
statistical comparison between groups, ANOVA was applied, and
2 analysis was used to compare gestation rates;
P 
0.05 was considered statistically significant.
Statistical analysis was carried out using the Statistical Package for
Social Sciences (SPSS, Inc., Chicago, IL).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Figure 1 shows the morphological
comparison by scanning electron microscopy of confluent monolayers of
primary cultures of EEC obtained from fertile patients (n = 10;
Fig. 1, A–C) and patients with IF (n = 10; Fig. 1, D–F) cultured
in the presence of a human blastocyst. Both EEC cultures were confluent
and healthy (Fig. 1, A and D). Cell membranes were covered with stubby
microvilli, and bulging of the membranes was also comparable (Fig. 1, B
and C, and E and F) between fertile and IF patients.
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Scanning electron microscopy comparison of confluent monolayers
from primary cultures of EEC obtained from fresh (n = 6) (Fig. 3, A and B) and frozen (n = 6; Fig. 3, C and D) endometria is shown in Fig. 3. Both EEC cultures appeared
similar. They were confluent with healthy-looking, flat, and elongated
cells; the plasma membranes were covered with short stubby microvilli,
and retraction fibers were common.
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In IF patients undergoing IVF, 496 blastocysts were obtained from
a total of 1240 day 2 embryos cocultured with AEEC (49.2% blastocyst
rate). In IF patients undergoing ovum donation, of a total of 544 day 2
embryos that initiated AEEC coculture, 332 human blastocysts (61.2% of
blastocyst development) were obtained. Blastocysts were transferred
back to the patient’s uterus on day 6 after oocyte retrieval. On day
6, blastocysts were transferred in the early (Fig. 5A), cavitated (Fig. 5B), expanded (Fig. 5C), or hatching (Fig. 5D) stage. Hatching and expanded blastocysts
were preferentially selected for transfer. Interestingly, hatching and
zona escape (Fig. 5D) were preceded by blastocyst expansion (Fig. 6B) and retraction (Fig. 6, C and D).
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The comparison of IVF and ovum donation cycles in patients with IF
undergoing AEEC and transfer at the blastocyst stage compared to those
in patients undergoing day 2 transfer, in terms of number of patients,
number of cycles, number of previous failed cycles, age and infertility
etiology, is presented in Table 1
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In 80 oocyte donation cycles, 6.8 ± 0.3 embryos/cycle started
coculture, resulting in 60.1% blastocyst formation; 2.3 ± 0.1
blastocysts were transferred, and 38 pregnancies were obtained
(implantation and pregnancy rates were 32.7% and 54.5%,
respectively). Eight miscarriages (21%), 18 ongoing pregnancies, and
12 live births were recorded. Nine cycles were canceled (12.5%) due to
embryonic development failure. In patients with IF undergoing 2-day
embryo transfer, implantation and pregnancy rates were significantly
lower compared to those in patients undergoing blastocyst transfer
(4.5% and 13.3%, respectively; P < 0.01; Table 2).
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). Patients
with IF undergoing IVF and day 2 embryo transfer had similar
implantation and pregnancy rates (10.7% and 35%, respectively; Table 3).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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When coculture systems are employed, in addition to the goal of high yield production of viable blastocysts and pregnancies, there are other important issues to be considered as end points, such as medical, ethical, and practical feasibility. For instance, sequential coculture with tubal-endometrial epithelium has proven efficient (10); tubal epithelium, however, is not routinely available and, therefore, not practical. The use of nonhuman cell lines always raises medical concerns because of the presence of unknown pathogens (viruses or prions) that could be identified in the future. Finally, ethical concerns can always be argued when tissues other than those from the same patient, in whom embryonic transfer has to be performed, are used for embryonic coculture. Only the use of AEEC from a previous cycle totally eliminates the risk of exogenous known or unknown bacterial or viral infection, thus overcoming medical, ethical, and practical concerns.
There are some reports concerning the coculture of human embryos on endometrial stromal cells; Jayot et al. (9) reported 90 cycles with coculture of embryos on stromal-epithelial monolayers obtained after 1 month of subculture and transfer at morula stage (day 4). They reported a pregnancy rate of 21% vs. 8% in previous cycles. Similar results were obtained by Prapas et al. (34) on a small sample using endometrial stromal cells and transferring on day 3. Also, cryopreserved endometrial epithelial cells have been used to influence fertilization and early cleavage with negative results (35). The rationale for using AEEC to grow human embryos until the blastocyst stage was based not only on the improvement of the blastocyst rate, but also on the induction of embryonic paracrine molecule secretion (27), which, in turn, regulates EEC adhesion molecules such as ß3 integrin subunit, which will improve uterine receptivity (28).
We do not know whether AEEC coculture is more or less efficient than sequential media for blastocyst development, as this was not the objective of our work. We are currently performing such a comparison within the same cohort of embryos from the same patient in a collaborative study. Also, the comparison of blastocyst transfer obtained after AEEC coculture or sequential media deserves further studies.
Clinical arguments for proposing AEEC coculture and transfer at the blastocyst stage are multiple: 1) in patients with implantation failure by allowing the embryonic genome expression, selecting therefore the best embryos together with a better synchrony between embryo and endometrial development; 2) to reduce the number of transferred embryos, avoiding multiple pregnancies in patients; and 3) to facilitate the embryonic development required for embryo biopsy and genetic screening for X- or Y-linked diseases or single gene alterations.
Our study design facilitates the dissecting out of the contributions of endometrial and embryonic factors to the implantation process by comparing the efficacy of blastocyst transfer on fixed days in different models of uterine receptivity. Ovum donation is an optimal model of uterine receptivity, because the endometrium of the recipient is artificially prepared by sequential administration of estrogen and P in physiological levels (33, 36). Ovulation induction drugs used in IVF induce a suboptimal receptivity model because of the induction of supraphysiological levels of steroids, which, in turn, produce morphological (37, 38) and biochemical (39) endometrial alterations relevant to uterine receptivity.
We have achieved a clear improvement in implantation rates in ovum donation patients with IF: 32.7% (optimal uterine receptivity/blastocyst) vs. 4.5% (optimal uterine receptivity/day 2 embryos). These differences were not obvious when patients with induction of ovulation were considered, 11.9% (suboptimal uterine receptivity/blastocyst) vs. 10.7% (suboptimal uterine receptivity/day 2 embryos). Data are not conclusive, but these figures suggest that in patients with optimal uterine receptivity, the improvement of the embryonic factor makes an important difference, whereas when uterine receptivity is suboptimal, the amelioration of the embryonic factor makes almost no contribution to the implantation process, indicating that we are lacking a key element, which is the understanding and improvement of uterine receptivity in IVF patients. A pitfall usually obtained from embryological studies is the consideration that implantation is merely the result of good embryonic quality. Although this is true, it is not the whole truth, because the endometrial factor is the real limiting factor, as demonstrated in this study.
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Footnotes |
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Received July 16, 1998.
Revised January 4, 1999.
Revised April 6, 1999.
Accepted April 14, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, IL-1ß and IL-1ra: IL-1 endometrial
microenvironment of the human embryo at the apposition phase under
physiological and supraphysiological steroid level conditions. J Reprod
Immunol. 31:165–184.[CrossRef][Medline]
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