The continuum of high ovarian response: a rational approach to the management of high responder patient subgroups

<div><p></p><p>Ovarian follicular responsiveness to controlled ovarian hyperstimulation (COH) with gonadotropins is extremely variable between individual patients, and even from cycle to cycle for the same patient. High responder patients are characterized by an exaggerated response to gonadotropin administration, accompanied by a higher risk for ovarian hyperstimulation syndrome (OHSS). In spite of its importance, the literature regarding high responders is characterized by heterogeneous classification methodologies. A clear separation should be drawn between risk factors for a high ovarian response and the actual response exhibited by a patient to stimulation. Similarly, it is important to distinguish between high ovarian response and development of clinically significant OHSS. In this article we: (1) review recent publications pertaining to the identification and clinical management of high responders, (2) propose an integrated clinical model to differentiate sub-groups within this population based on this review, and (3) suggest specific protocols for each sub-group. The model is based on a chronological patient assessment in an effort to target treatment based on the specific clinical circumstances. It is our hope that the algorithm we have developed will assist clinicians to supply targeted and precise treatments in order to achieve a favorable reproductive outcome with minimum complications for each patient.</p></div

The elusive MAESTRO gene: Its human reproductive tissue-specific expression pattern

<div><p>The encoded transcript of the Maestro—<i>Male-specific Transcription in the developing Reproductive Organs</i> (MRO) gene exhibits sexual dimorphic expression during murine gonadal development. The gene has no homology to any known gene and its expression pattern, protein function or structure are still unknown. Previously, studying gene expression in human ovarian cumulus cells, we found increased expression of <i>MRO</i> in lean-type Polycystic Ovarian Syndrome (PCOS) subjects, as compared to controls. In this study, we examined the <i>MRO</i> splice variants and protein expression pattern in various human tissues and cells. We found a differential expression pattern of the <i>MRO</i> 5’-UTR region in luteinized granulosa-cumulus cells and in testicular tissues as compared to non-gonadal tissues. Our study also shows a punctate nuclear expression pattern and disperse cytoplasmic expression pattern of the MRO protein in human granulosa-cumulus cells and in testicular germ cells, which was later validated by western blotting. The tentative and unique features of the protein hampered our efforts to gain more insight about this elusive protein. A better understanding of the tissue-specific <i>MRO</i> isoforms expression patterns and the unique structure of the protein may provide important insights into the function of this gene and possibly to the pathophysiology of PCOS.</p></div

Clinical parameters of PCOS and control patients.

<p>Clinical parameters of PCOS and control patients.</p

List of PCR primers.

<p>List of PCR primers.</p

<i>MRO</i> expression in human ovarian sections.

<p>Immunofluorescence was performed on non-PCOS (A) and PCOS (B and C) ovarian sections using <i>the</i> FL-248 antibody (4ug/ml). Nuclear staining is apparent in the granulosa cells (magnified images), while the blocking peptide abolished the MRO signal (C). <i>MRO</i> expression was observed in green (Alexa-fluor 488) and nucleus in blue (DAPI). Magnification: x100. Duration of exposure: 2000 milliseconds (non-PCOS) and 1500 milliseconds (PCOS) demonstrating that even with short exposure the fluorescence intensity is higher in PCOS samples.</p

Relative expression of <i>MRO</i> transcripts was analyzed by qPCR.

<p>(A) Schematic representation of the <i>MRO</i> splice variants and the position of the TaqMan probes. Overall expression detected by the pQ8/9 probe. pQ1/3 spans exons 1–3 and detects the 'b' isoforms (<i>MROb</i>1-4); pQ2/3 spans exons 2/3 and detects isoforms <i>MROa</i>,c,d. The expression of <i>MRO</i> was normalized to 18s RNA and presented as relative a expression ratio (1/ΔCt). (B) <i>MRO</i> transcripts analysis in GCs and CCs from PCOS and control subjects. The overall expression, shown by exon 8/9 was higher in PCOS cells vs that of controls (* in GCs and * in CCs; p ≤0.05). Inverse expression of exon 1/3 in GCs and CCs vs exon 2/3 was detected. (C) Inverse expression of exon 2/3 vs 1/3 in kidney, liver, brain and post-menopausal ovary.</p

<i>MRO</i> expression in human tissues with FL-248.

<p><i>MRO</i> expression in (A) ovarian tissue of a post-menopausal subject with <b>no active folliculogenesis</b> (age 52); (B) adult normal testis; (C) Typical testicular seminoma; (D) Cerebrum (brain) tissue. Abbreviations: GLC–granulosa-lutein cells, TL–theca lutein cells, SC–Sertoli cells, SG–spermatogonia, PS–primary spermatocyte, SP–spermatids, NP–Neuropil, GL–glial cells. Bar ruler, 100um.</p

Summary of the MRO splice variants detected in GCs, CCs and human tissues by standard PCR and TaqMan qPCR analysis.

<p>(+) indicates positive detection and intensity (++++ being the highest). (-) indicates no product detected.</p

MRO is detected in both the cytoplasm and nucleus by western Immunoblot.

<p>A) Nuclear and cytoplasmic extracts were prepared from purified GCs, electrophoresed through a 4–12% reducing SDS-PAGE gel, transferred to 0.2um nitrocellulose, blocked, and probed with the MRO FL-248 (Santa Cruz), anti-HDAC1 (Abcam) and anti-Actin (Sigma-Aldrich) antibodies. MRO (~28kDa) is found at similar abundance in both the cytoplasmic and nuclear fraction (top panel). HDAC1 served as a nuclear specific control to ensure the purity of the extract (middle panel) and Actin served as a loading control (bottom panel). B) Protein extracts from portions of GCs from the different isolation method were resolved. GCs pre-wash (Load), after the 1^st wash (post-wash) and after the Ficoll gradient (Post-Ficoll) were probes with the lymphocyte marker anti ἀCD45. Actin served as a loading control (bottom panel).</p

Schematic representation of the human <i>Maestro (MRO)</i> gene structure.

<p>The start codon of the Open Reading Frame (ORF) indicated by an arrow is localized in exon 2 for <i>MRO</i>a, <i>MRO</i>a2, <i>MRO</i>c, <i>MRO</i>d, and in exon 4 for ‘<i>MRO</i>b’ (b1-b4) transcript variants. <i>MRO</i>a (NM_031939.3), <i>MRO</i>d (NM_001127176.1), and <i>MRO</i>c (NM_001127175.1) coding regions consist of 753bp, 648bp, and 492bp and encode 248aa, 262aa, and 210aa protein, respectively. <i>MRO</i>b1 (NM_001127174), <i>MRO</i>b2, and <i>MRO</i>b3 consist of 678bp, 827bp, and 792bp and encode 196aa, 248aa, and 210aa proteins, respectively. <i>MRO</i>a is considered as a non-sense mediated decay (NMD) transcript. The ‘a’ variant differs by a 103bp additional 5’ exon (exon 4) compared to the ‘c’ and ‘d’ variants. The ‘c’ variant lacks one in-frame exon (exon 7) compared to ‘d’. The <i>MRO</i> ‘b’ variants (<i>MRO</i>b1-4) contain a distinct 5' UTR differ from <i>MRO</i>a, c, d by a 76bp non-coding exon (exon 1), and differ from each other by one alternate exon (exon 3) and one in-frame exon (exon 7). <i>MRO</i>b4 (BC029860.1) is the longest transcript, consisting of 948bp and encoding 248aa. The predictive protein isoforms have 2 distinct N-terminus with expected sizes of 196 aa– 248 aa and a mass of 23-29kDda (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0174873#pone.0174873.s001" target="_blank">S1 Fig</a>).</p