Poor Metabolic Quality Embryo is Capable to Implant – A Case Report

Volume 3 | Issue 2 Introduction Poor Metabolic Quality Embryo is Capable to Implant – A Case Report Bartmann AK*1,2,3, Silva LFI1,4, Silva LLM2, Mello DFRE2, Gualberto JR2, Bertoncelo DM2, Sanchez IG2, Millani KVS2 and Contini SHT3 1Human Reproduction Center of the Ana Bartmann Clinic, Brazil 2Medical School, University of Ribeirão Preto (UNAERP), Brazil 3Biotechnology Department,University of Ribeirão Preto (UNAERP), Brazil 4Department of Obstetrics and Gynecology, School of Medicine of Ribeirão Preto, University of São Paulo (USP), Brazil *Corresponding author: Bartmann AK, Electro Bonini Hospital University of Ribeirão Preto (UNAERP), Av Leao XI, s / n CEP: 14096900, Ribeirao Preto SP, Brazil, Tel: 00551639415121, E-mail: anabartmann@uol. com.br Citation: Bartmann AK, Silva LFI, Silva LLM, Mello DFRE, Gualberto JR, et al. (2017) Poor Metabolic Quality Embryo is Capable to Implant – A Case Report. J Gynecol Res 3(2): 201

Increased amounts of lactate are synthesized in embryo development [9]. Research suggests that high lactate production by embryos in their pre-implantation stage is an adaptation to culture conditions and the higher the production the better the adaptive conditions. Gardner et al. 2001 [10] -suggest that the differences in the use of pyruvate or lactate at different stages of development are due to differences in the regulation of lactate dehydrogenase (LDH). This enzyme catalyzes the lactate oxidation by a NAD+ molecule, leading to the formation of pyruvate and NADH and vice versa, allowing the cell to obtain pyruvate from a single reaction, simpler than the series of reactions required for glycolysis.
In this article, we report a case of a 33-year-old patient, married to a 29-year-old man, who underwent controlled ovarian hyperstimulation (COH) with standard protocol and ICSI [11]. Recombinant FSH therapy (Gonal-F, Serono Pharmaceuticals, Boulogne, France) was initiated at a dosage of 225 IU/day for 10 days, until the day of hCG administration (Ovidrel, Organon Pharmaceuticals, Saint-Denis, France). Administration of hCG was performed as soon as ≥4 pre-ovulatory follicles (16-22 mm in diameter) were observed. Oocyte retrieval was performed 34 hours after trigger. We collected 6 oocytes from 7 follicles. All oocytes fertilized and became morphologically normal embryos.
This being the case, we conclude that the greater the metabolic capacity of an embryo the better its quality. Accordingly, when we analyze its biochemistry, an embryo with high metabolic capacity must a priori present a low concentration of pyruvate and a high concentration of lactate.
The objective of this paper is to report the case of a low metabolic embryo implantation.

Case presentation
Since she did not have endometrium thickness >8mm, we decided to freeze all embryos for further transfer. Patient had no endometrial phatology or systemic disease. The cause of infertility was male factor (moderate oligospermia).

Materials and Methods
At the end of COH, six oocytes were collected and the ICSI procedure was performed, resulting in six embryos. The embryos were cultured in individual microdroplets of a Global Life Total® (Life Global®, Guilford-CT, USA) medium for three days without washing. On the first day of culture (D1), a falcon 353002 dish was prepared with six side microdroplets and a central microdroplet with 40 uL of Life Global Total ® supplemented medium, covered with 5 mL of mineral oil (Irvine Scientific -Santa Ana, CA). The embryos were cultured within the six microdroplets. The central microdroplet remained without embryo culture for three days (control droplet). In the morning of the third day (D3), the embryos were photographed and morphologically assessed according to Veeck's criteria [12]. They were placed in a new culture dish. The microdroplets of the previously cultured dish were separately collected after complete removal of mineral oil. We analyzed them by evaluating their pyruvate and lactate levels, first in the central droplet (without embryo culture) and subsequently in the other microdroplets where culture took place. We compared the metabolic levels of the central microdroplet and of the microdroplets that had had culture, and evaluated the consumption and production of the metabolites found, establishing a metabolic profile of each embryo. As the patient had thin endometrium, all embryos were thawed for further intra-uterine transfer.
In the first transfer after endometrial preparation, two embryos did not survive thawing and two others were transferred, but did not result in gestation.
The clinical monitoring of lactate and pyruvate by gas chromatography technique coupled with mass spectrometry (GC/MS) required a prior derivatization of the sample; therefore we added 1 mL of ethanol to 30 µL of medium followed by centrifugation. The supernatant was transferred to a glass vial and evaporated. The material was redissolved in 50µL of pyridine, 50µL of the silanizing reagent BSTFA (bis(trimethylsilyl)trifluoroacetamide -SUPELCO), 20.5µl of TMCS (98 % (Chloromethyl)trimethylsilane -ALDRICH), and heated to 50 °C for 15 minutes [14]. After silanization, we dosed these metabolites concentrations in the culture medium by GC/MS technique [15]. In the first transfer after endometrial preparation, two embryos did not survive thawing and two others were transferred, but did in the Krebs cycle [6]. Pyruvate is mainly consumed by the embryo at the morula stage [7,8]. Moreover, pyruvate is the only source of energy used by the embryo in its first mitotic division and for this reason it is essential for its development during the period that immediately follows fertilization [5].

Journal of Gynecology Research
Results not result in gestation. In the second transfer after a new endometrial preparation, the two remaining embryos were transferred, resulting in a single gestation. The transfers were performed after standard endometrial preparation with the use of estrogen and subsequent maintenance of the endometrium with progesterone. The two transfers were performed by the same physician, with endometrial thickness greater than 8 mm.
The research was carried out at the Human Reproduction Center of the Ana Bartmann Clinic in partnership with the University of Ribeirão Preto (UNAERP), between August 2014 and June 2015. This case is part of a group of patients from a prospective controlled research that is currently underway. The ethical approval protocol was approved by the medical ethic committee of the hospital. Each couple whose embryos were enrolled into the study signed a written informed consent. Of the six embryos obtained by ICSI procedure (Table 1 and Figure 1), there was: a grade 8A embryo with pyruvate uptake of 10.43% and lactate production of 2.5% (compared against the control droplet) that did not survive heating. A grade 8B embryo with pyruvate uptake of 10.54% and lactate production of 3.11% (against the control droplet), that also did not survive heating. A grade 8B embryo with pyruvate uptake of 9.91% and lactate production of 0.84% (against the control droplet). A grade 8B embryo with pyruvate uptake of 8.81% and lactate production of 0.92%, (against the control). These last two were transferred, but did not result in clinical pregnancy. A grade 6B embryo with pyruvate uptake of 8.41% and lactate production of 0.00% (against the control droplet). A grade 7B embryo with pyruvate uptake of 8.75% and lactate production of 0.39% (against the control droplet). These two last embryos were transferred and resulted in a single-fetus pregnancy.

Discussion
Although it is not possible to draw conclusions based on a single case report, we may notice that embryos with high pyruvate uptake and lactate production showed an inverse relationship between implantation potential and clinical pregnancy. Additional studies are required to validate these observations, but some considerations must be made. The first is related to the concept of "best embryo". Studies with a "time lapse" methodology, for example, have shown that embryos with high developmental speed have greater implantation potential [16]. It is not known, however, if high metabolic profile is related to greater implantation rates or greater cellular distress, which can influence the development of embryos [17]. A low metabolic rate, therefore, could be a positive characteristic in terms of response to the stress of cryopreservation [18].

Conclusion
The excess of nutrients as well as their lack may be harmful to the development of embryos. Thus, it is of the utmost importance to understand the metabolic basis of pre-implantational stage in order to reduce the stress to which embryos are submitted during IVF procedures.
Besides the importance of evaluating the metabolic rate of the embryo, it is known that the study of its genetic is an inherent condition for implantation success [19] Corroborating these data, the epigenetic plays an important role in embryo cellular signaling as well as in embryo programming in response to environmental disturbances [20].
It is important to recognize that there are many variables, which can play a role in human embryo implantation and puzzle the interpretation of data. This means that it is soon to state that a good embryo has high metabolic rate. More studies are needed to establish what a good embryo mean in terms of metabolism. It is possible that the rates of pyruvate uptake and lactate production do not need to be as high as theory suggests. Or that the proportions between metabolites are more important than their isolated concentrations.
We conclude that the metabolic rate analyzed alone is not a good predictor of implantation, since, as we showed, embryos with poor metabolic rate are also capable to implant.

Consent
A written informed consent was obtained from the patient for the publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.