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. 2019 Jul;36(7):1457-1469.
doi: 10.1007/s10815-019-01489-8. Epub 2019 Jun 11.

Cumulus cell pappalysin-1, luteinizing hormone/choriogonadotropin receptor, amphiregulin and hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 1 mRNA levels associate with oocyte developmental competence and embryo outcomes

Richard J Kordus  1   2 , Akhtar Hossain  3 , Michael C Corso  1 , Hrishikesh Chakraborty  4 , Gail F Whitman-Elia  5   6 , Holly A LaVoie  7
Affiliations

Cumulus cell pappalysin-1, luteinizing hormone/choriogonadotropin receptor, amphiregulin and hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 1 mRNA levels associate with oocyte developmental competence and embryo outcomes

Richard J Kordus et al. J Assist Reprod Genet. 2019 Jul.

Abstract

Purpose: To determine whether a selected set of mRNA biomarkers expressed in individual cumulus granulosa cell (CC) masses show association with oocyte developmental competence, embryo ploidy status, and embryo outcomes.

Methods: This prospective observational cohort pilot study assessed levels of mRNA biomarkers in 163 individual CC samples from 15 women stimulated in antagonist cycles. Nineteen mRNA biomarker levels were measured by real-time PCR and related to the development of their corresponding individually cultured oocytes and subsequent embryos, embryo ploidy status, and live birth outcomes.

Results: PAPPA mRNA levels were significantly higher in CC from oocytes that led to euploid embryos resulting in live births and aneuploid embryos compared to immature oocytes by ANOVA. LHCGR mRNA levels were significantly higher in CC of oocytes resulting in embryos associated with live birth compared to immature oocytes and oocytes resulting in arrested embryos by ANOVA. Using a general linearized mixed model to assess ploidy status, CC HSD3B mRNA levels in oocytes producing euploid embryos were significantly lower than other oocyte outcomes, collectively. When transferred euploid embryos outcomes were analyzed by ANOVA, AREG mRNA levels were significantly lower and PAPPA mRNA levels significantly higher in CC from oocytes that produced live births compared to transferred embryos that did not form a pregnancy.

Conclusions: Collectively, PAPPA, LHCGR, and AREG mRNA levels in CC may be able to identify oocytes with the best odds of resulting in a live birth, and HSD3B1 mRNA levels may be able to identify oocytes capable of producing euploid embryos.

Keywords: Cumulus cells; Euploid embryo; Oocyte developmental competence; Real-time PCR; mRNA levels.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Fig. 1
Fig. 1
Flow diagram summarizing the study population from oocyte retrieval to final outcome. Oocytes (164) were retrieved from 15 patients and individually cultured. Individual CC masses from each oocyte were collected and mRNA was harvested from each mass. Of the 164 oocytes, 134 were mature and 30 were immature. All 134 were injected with sperm. Seventy-three of the fertilized oocytes became blastocysts and were biopsied for PGT-A testing and all were vitrified. PGT-A results indicated that 39 embryos were euploid and 34 were aneuploid. Nineteen of the euploid embryos were transferred and 20 remained in cryostorage. Ten embryos from 7 patients failed to implant while 9 embryos from 8 patients implanted and resulted in live births. *The control TBP mRNA did not amplify in one CC sample from an oocyte yielding a euploid embryo and was not included in the data analysis. RNA analyses were performed on n = 163 individual cumulus masses
Fig. 2
Fig. 2
Principal component analysis biplot for CC mRNA expression. Biplot of the log2-transformed mRNA levels for 7 genes from 14 patients along the first and second principal components (PC1 and PC2). PC1 accounted for 35.7% of the observed variance while PC2 accounted for 22.9% of the variance. Each data point represents an individual CC mass mRNA level associated with an individual oocyte (n = 151 oocytes). The biplot demonstrates the relationship between individual CC mRNA level patterns and the correlation between the different genes. The closer the data points are to each other, the more similar the normalized CC gene expression patterns. The closer the arrows are to each other, the higher the correlation between the normalized CC gene expressions. Groups represent CC mRNA from oocytes with the following descriptions: aneuploid = mature oocytes resulting in aneuploid embryos; arrested = mature oocytes resulting in embryos that did not reach the blastocyst stage; euploid = mature oocytes resulting in euploid blastocysts that were not transferred; failed fert = mature oocytes that did not fertilize; immature = immature oocytes that were not fertilized; live birth = oocytes that resulted in euploid embryos that resulted in live births; no pregnancy = oocytes that resulted in euploid embryos that did not result in a pregnancy
Fig. 3
Fig. 3
Biomarkers for CC mRNA expression associated with mature oocyte competence and embryo outcomes. To determine the differences in CC mRNA between groups where oocytes had different developmental and embryo outcomes, target mRNA levels were compared using repeated measures ANOVA followed by Tukey’s post hoc test (adjusted P values) for pairwise comparisons. Groups represent CC mRNA from oocytes with the following descriptions: aneuploid = mature oocytes resulting in aneuploid embryos; arrested = mature oocytes resulting in embryos that did not reach the blastocyst stage; failed fert = mature oocytes that did not fertilize; immature = immature oocytes that were not fertilized; live birth = oocytes that resulted in transferred euploid embryos that resulted in live births; no pregnancy = oocytes that resulted in transferred euploid embryos that did not result in a pregnancy. Data are presented as the median copy number (line inside box), first and third quartile (bottom and top of box), and highest and lowest data points (top and bottom of whiskers). Groups with different letters (a and b) exhibit significant differences (P < 0.05)
Fig. 4
Fig. 4
Biomarkers from the GLMM model associated with oocytes producing euploid embryos versus mature oocytes with other outcomes. To evaluate CC biomarker mRNA level association with oocytes capable of producing euploid embryos, a model was fit with 78 CC samples from 11 patients and included the biomarkers: CYP11A1, CYP19A1, HSD3B, IGFBP5, PAPPA, PGR, PGRMC1, ING1, LHCGR, and STARD1. Higher HSD3B mRNA level significantly decreased the odds of an oocyte resulting in a euploid embryo (OR = 0.408, 95% CI 0.175 to 0.953). Higher ING1 mRNA levels marginally decreased the odds of an oocyte resulting in a euploid embryo (OR = 0.552, 95% CI 0.297 to 1.027). Groups represent CC mRNA from oocytes with the following descriptions: abnormal = mature oocytes resulting in aneuploid blastocysts, oocytes producing embryos that did not reach the blastocyst stage, and mature oocytes that failed to fertilize; euploid = mature oocytes that resulted in euploid embryos regardless of whether they were transferred or remained vitrified. Data are presented as stated in Fig. 3 legend
Fig. 5
Fig. 5
CC biomarkers associated with oocytes giving rise to euploid embryos with live birth or no pregnancy. Transferred embryos (n = 19) from all 15 patients were assessed for mRNA level differences between those oocytes yielding embryos that resulted in a live birth and those that did not form a pregnancy using repeated measures ANOVA followed by Tukey’s post hoc test (adjusted P values) for pairwise comparisons. Of the transferred embryos, 9 resulted in live births. AREG mRNA levels were significantly lower in CCs from oocytes that resulted in live births (n = 8 from 8 patients) compared to the no pregnancy group (n = 7 from 5 patients) (P < 0.05). PAPPA mRNA expression was significantly increased in CCs from oocytes producing embryos that resulted in live births (n = 9 from 8 patients) compared to no pregnancy (n = 10 from 7 patients) (P < 0.05). GREM1 mRNA levels (n = 3/group) were not significantly different and not shown. Data are presented as in Fig. 3 legend
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